RESUMO
Sustained energy starvation leads to activation of AMP-activated protein kinase (AMPK), which coordinates energy status with numerous cellular processes including metabolism, protein synthesis, and autophagy. Here, we report that AMPK phosphorylates the histone methyltransferase EZH2 at T311 to disrupt the interaction between EZH2 and SUZ12, another core component of the polycomb repressive complex 2 (PRC2), leading to attenuated PRC2-dependent methylation of histone H3 at Lys27. As such, PRC2 target genes, many of which are known tumor suppressors, were upregulated upon T311-EZH2 phosphorylation, which suppressed tumor cell growth both in cell culture and mouse xenografts. Pathologically, immunohistochemical analyses uncovered a positive correlation between AMPK activity and pT311-EZH2, and higher pT311-EZH2 correlates with better survival in both ovarian and breast cancer patients. Our finding suggests that AMPK agonists might be promising sensitizers for EZH2-targeting cancer therapies.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Animais , Carcinogênese/genética , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Epigênese Genética , Feminino , Histonas/metabolismo , Humanos , Camundongos , Proteínas de Neoplasias , Proteínas Nucleares/metabolismo , Oncogenes , Neoplasias Ovarianas/metabolismo , Fosforilação , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/fisiologia , Fatores de Transcrição , Regulação para CimaRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper. The original Letter has not been corrected.
RESUMO
Treatments that target immune checkpoints, such as the one mediated by programmed cell death protein 1 (PD-1) and its ligand PD-L1, have been approved for treating human cancers with durable clinical benefit. However, many patients with cancer fail to respond to compounds that target the PD-1 and PD-L1 interaction, and the underlying mechanism(s) is not well understood. Recent studies revealed that response to PD-1-PD-L1 blockade might correlate with PD-L1 expression levels in tumour cells. Hence, it is important to understand the mechanistic pathways that control PD-L1 protein expression and stability, which can offer a molecular basis to improve the clinical response rate and efficacy of PD-1-PD-L1 blockade in patients with cancer. Here we show that PD-L1 protein abundance is regulated by cyclin D-CDK4 and the cullin 3-SPOP E3 ligase via proteasome-mediated degradation. Inhibition of CDK4 and CDK6 (hereafter CDK4/6) in vivo increases PD-L1 protein levels by impeding cyclin D-CDK4-mediated phosphorylation of speckle-type POZ protein (SPOP) and thereby promoting SPOP degradation by the anaphase-promoting complex activator FZR1. Loss-of-function mutations in SPOP compromise ubiquitination-mediated PD-L1 degradation, leading to increased PD-L1 levels and reduced numbers of tumour-infiltrating lymphocytes in mouse tumours and in primary human prostate cancer specimens. Notably, combining CDK4/6 inhibitor treatment with anti-PD-1 immunotherapy enhances tumour regression and markedly improves overall survival rates in mouse tumour models. Our study uncovers a novel molecular mechanism for regulating PD-L1 protein stability by a cell cycle kinase and reveals the potential for using combination treatment with CDK4/6 inhibitors and PD-1-PD-L1 immune checkpoint blockade to enhance therapeutic efficacy for human cancers.
Assuntos
Antígeno B7-H1/metabolismo , Proteínas Culina/metabolismo , Ciclina D/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Vigilância Imunológica , Neoplasias/imunologia , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Evasão Tumoral/imunologia , Proteínas 14-3-3/metabolismo , Animais , Antígeno B7-H1/biossíntese , Proteínas Cdh1/metabolismo , Ciclo Celular , Linhagem Celular , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Feminino , Humanos , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Masculino , Camundongos , Proteínas Nucleares/química , Fosforilação , Receptor de Morte Celular Programada 1/metabolismo , Neoplasias da Próstata/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , Proteínas Repressoras/químicaRESUMO
The mechanistic target of rapamycin (mTOR) has a key role in the integration of various physiological stimuli to regulate several cell growth and metabolic pathways. mTOR primarily functions as a catalytic subunit in two structurally related but functionally distinct multi-component kinase complexes, mTOR complex 1 (mTORC1) and mTORC2 (refs 1, 2). Dysregulation of mTOR signalling is associated with a variety of human diseases, including metabolic disorders and cancer. Thus, both mTORC1 and mTORC2 kinase activity is tightly controlled in cells. mTORC1 is activated by both nutrients and growth factors, whereas mTORC2 responds primarily to extracellular cues such as growth-factor-triggered activation of PI3K signalling. Although both mTOR and GßL (also known as MLST8) assemble into mTORC1 and mTORC2 (refs 11, 12, 13, 14, 15), it remains largely unclear what drives the dynamic assembly of these two functionally distinct complexes. Here we show, in humans and mice, that the K63-linked polyubiquitination status of GßL dictates the homeostasis of mTORC2 formation and activation. Mechanistically, the TRAF2 E3 ubiquitin ligase promotes K63-linked polyubiquitination of GßL, which disrupts its interaction with the unique mTORC2 component SIN1 (refs 12, 13, 14) to favour mTORC1 formation. By contrast, the OTUD7B deubiquitinase removes polyubiquitin chains from GßL to promote GßL interaction with SIN1, facilitating mTORC2 formation in response to various growth signals. Moreover, loss of critical ubiquitination residues in GßL, by either K305R/K313R mutations or a melanoma-associated GßL(ΔW297) truncation, leads to elevated mTORC2 formation, which facilitates tumorigenesis, in part by activating AKT oncogenic signalling. In support of a physiologically pivotal role for OTUD7B in the activation of mTORC2/AKT signalling, genetic deletion of Otud7b in mice suppresses Akt activation and Kras-driven lung tumorigenesis in vivo. Collectively, our study reveals a GßL-ubiquitination-dependent switch that fine-tunes the dynamic organization and activation of the mTORC2 kinase under both physiological and pathological conditions.
Assuntos
Carcinogênese , Endopeptidases/metabolismo , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Endopeptidases/deficiência , Endopeptidases/genética , Ativação Enzimática , Feminino , Homeostase , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/química , Fosforilação , Poliubiquitina/metabolismo , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/biossíntese , Serina-Treonina Quinases TOR/química , Homólogo LST8 da Proteína Associada a mTORRESUMO
The ERG gene is fused to TMPRSS2 in approximately 50% of prostate cancers (PrCa), resulting in its overexpression. However, whether this is the sole mechanism underlying ERG elevation in PrCa is currently unclear. Here we report that ERG ubiquitination and degradation are governed by the Cullin 3-based ubiquitin ligase SPOP and that deficiency in this pathway leads to aberrant elevation of the ERG oncoprotein. Specifically, we find that truncated ERG (ΔERG), encoded by the ERG fusion gene, is stabilized by evading SPOP-mediated destruction, whereas prostate cancer-associated SPOP mutants are also deficient in promoting ERG ubiquitination. Furthermore, we show that the SPOP/ERG interaction is modulated by CKI-mediated phosphorylation. Importantly, we demonstrate that DNA damage drugs, topoisomerase inhibitors, can trigger CKI activation to restore the SPOP/ΔERG interaction and its consequent degradation. Therefore, SPOP functions as a tumor suppressor to negatively regulate the stability of the ERG oncoprotein in prostate cancer.
Assuntos
Proteínas Nucleares/fisiologia , Neoplasias da Próstata/metabolismo , Proteínas Repressoras/fisiologia , Transativadores/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Proteínas Culina/metabolismo , Progressão da Doença , Etoposídeo/farmacologia , Células HEK293 , Humanos , Masculino , Dados de Sequência Molecular , Invasividade Neoplásica , Neoplasias da Próstata/patologia , Domínios e Motivos de Interação entre Proteínas , Proteólise , Regulador Transcricional ERG , Proteínas Supressoras de Tumor/fisiologiaRESUMO
While many disease-associated single nucleotide polymorphisms (SNPs) are associated with gene expression (expression quantitative trait loci, eQTLs), a large proportion of complex disease genome-wide association study (GWAS) variants are of unknown function. Some of these SNPs may contribute to disease by regulating gene splicing. Here, we investigate whether SNPs that are associated with alternative splicing (splice QTL or sQTL) can identify novel functions for existing GWAS variants or suggest new associated variants in chronic obstructive pulmonary disease (COPD). RNA sequencing was performed on whole blood from 376 subjects from the COPDGene Study. Using linear models, we identified 561,060 unique sQTL SNPs associated with 30,333 splice sites corresponding to 6,419 unique genes. Similarly, 708,928 unique eQTL SNPs involving 15,913 genes were detected at 10% FDR. While there is overlap between sQTLs and eQTLs, 55.3% of sQTLs are not eQTLs. Co-localization analysis revealed that 7 out of 21 loci associated with COPD (p<1x10-6) in a published GWAS have at least one shared causal variant between the GWAS and sQTL studies. Among the genes identified to have splice sites associated with top GWAS SNPs was FBXO38, in which a novel exon was discovered to be protective against COPD. Importantly, the sQTL in this locus was validated by qPCR in both blood and lung tissue, demonstrating that splice variants relevant to lung tissue can be identified in blood. Other identified genes included CDK11A and SULT1A2. Overall, these data indicate that analysis of alternative splicing can provide novel insights into disease mechanisms. In particular, we demonstrated that SNPs in a known COPD GWAS locus on chromosome 5q32 influence alternative splicing in the gene FBXO38.
Assuntos
Processamento Alternativo , Proteínas F-Box/genética , Estudo de Associação Genômica Ampla/métodos , Doença Pulmonar Obstrutiva Crônica/genética , Idoso , Idoso de 80 Anos ou mais , Arilsulfotransferase/genética , Quinases Ciclina-Dependentes/genética , Éxons , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Análise de Sequência de RNARESUMO
BACKGROUND: 3-phosphoinositide-dependent protein kinase-1 (PDK1) acts as a master kinase of protein kinase A, G, and C family (AGC) kinase to predominantly govern cell survival, proliferation, and metabolic homeostasis. Although the regulations to PDK1 downstream substrates such as protein kinase B (AKT) and ribosomal protein S6 kinase beta (S6K) have been well established, the upstream regulators of PDK1, especially its degrader, has not been defined yet. METHOD: A clustered regularly interspaced short palindromic repeats (CRISPR)-based E3 ligase screening approach was employed to identify the E3 ubiquitin ligase for degrading PDK1. Western blotting, immunoprecipitation assays and immunofluorescence (IF) staining were performed to detect the interaction or location of PDK1 with speckle-type POZ protein (SPOP). Immunohistochemistry (IHC) staining was used to study the expression of PDK1 and SPOP in prostate cancer tissues. In vivo and in vitro ubiquitination assays were performed to measure the ubiquitination conjugation of PDK1 by SPOP. In vitro kinase assays and mass spectrometry approach were carried out to identify casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3)-mediated PDK1 phosphorylation. The biological effects of PDK1 mutations and correlation with SPOP mutations were performed with colony formation, soft agar assays and in vivo xenograft mouse models. RESULTS: We identified that PDK1 underwent SPOP-mediated ubiquitination and subsequent proteasome-dependent degradation. Specifically, SPOP directly bound PDK1 by the consensus degron in a CK1/GSK3ß-mediated phosphorylation dependent manner. Pathologically, prostate cancer patients associated mutations of SPOP impaired PDK1 degradation and thus activated the AKT kinase, resulting in tumor malignancies. Meanwhile, mutations that occurred around or within the PDK1 degron, by either blocking SPOP to bind the degron or inhibiting CK1 or GSK3ß-mediated PDK1 phosphorylation, could markedly evade SPOP-mediated PDK1 degradation, and played potently oncogenic roles via activating the AKT kinase. CONCLUSIONS: Our results not only reveal a physiological regulation of PDK1 by E3 ligase SPOP, but also highlight the oncogenic roles of loss-of-function mutations of SPOP or gain-of-function mutations of PDK1 in tumorigenesis through activating the AKT kinase.
Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/metabolismo , Xenoenxertos , Humanos , Camundongos , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Proteólise , Proteínas Repressoras/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, can significantly improve the reprogramming efficiency of somatic cells. However, whether TSA has a detrimental effect on other kinds of embryos is largely unknown because of the lack of integrated analysis of the TSA effect on natural fertilized embryos. To investigate the effect of TSA on mouse embryo development, we analyzed preimplantation and post-implantation development of in vivo, in vitro fertilized, and parthenogenetic embryos treated with TSA at different concentrations and durations. In vivo fertilized embryos appeared to be the most sensitive to TSA treatment among the three groups, and the blastocyst formation rate decreased sharply as TSA concentration and treatment time increased. TSA treatment also reduced the livebirth rate for in vivo fertilized embryos from 56.59 to 38.33% but did not significantly affect postnatal biological functions such as the pups' reproductive performance and their ability for spatial learning and memory. Further analysis indicated that the acetylation level of H3K9 and H4K5 was enhanced by TSA treatment at low concentrations, while DNA methylation appeared to be also disturbed by TSA treatment only at high concentration. Thus, our data indicates that TSA has different effects on preimplantation embryonic development depending on the nature of the embryo's reproductive origin, the TSA concentration and treatment time, whereas the effect of TSA at the indicated concentration on postnatal function was minor.
Assuntos
Blastocisto/citologia , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Ácidos Hidroxâmicos/farmacologia , Aprendizagem/fisiologia , Memória/fisiologia , Reprodução , Acetilação , Animais , Animais Recém-Nascidos , Blastocisto/efeitos dos fármacos , Metilação de DNA , Implantação do Embrião , Transferência Embrionária , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Fertilização in vitro , Inibidores de Histona Desacetilases/farmacologia , Histonas/química , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Técnicas de Transferência Nuclear , GravidezRESUMO
Pre-implantation embryos undergo genome-wide DNA demethylation, however certain regions, like imprinted loci remain methylated. Further, the mechanisms ensuring demethylation resistance by TRIM28 in epigenetic reprogramming remain poorly understood. Here, TRIM28 was knocked down in oocytes, and its effects on porcine somatic cell nuclear transfer (SCNT) embryo development was examined. Our results showed that SCNT embryos constructed from TRIM28 knockdown oocytes had significantly lower cleavage (53.9 ± 3.4% vs 64.8 ± 2.7%) and blastocyst rates (12.1 ± 4.3% vs 19.8 ± 1.9%) than control-SCNT embryos. The DNA methylation levels at the promoter regions of the imprinting gene IGF2 and H19 were significantly decreased in the 4-cell stage, and the transcript abundance of other imprinting gene was substantially increased. We also identified an aberrant two-fold decrease in the expression of CXXC1and H3K4me3 methyltransferase (ASH2L and MLL2), and the signal intensity of H3K4me3 had a transient drop in SCNT 2-cell embryos. Our results indicated that maternal TRIM28 knockdown disrupted the genome imprints and caused epigenetic variability in H3K4me3 levels, which blocked the transcription activity of zygote genes and affected the normal developmental progression of porcine SCNT embryos.
Assuntos
Blastocisto/citologia , Desenvolvimento Embrionário , Epigênese Genética , Fertilização in vitro/veterinária , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Transferência Nuclear/veterinária , Animais , Blastocisto/metabolismo , Metilação de DNA , Feminino , Genoma , SuínosRESUMO
Cullin 3-RING ligases (CRL3) play pivotal roles in the regulation of various physiological and pathological processes, including neoplastic events. The substrate adaptors of CRL3 typically contain a BTB domain that mediates the interaction between Cullin 3 and target substrates to promote their ubiquitination and subsequent degradation. The biological implications of CRL3 adaptor proteins have been well described where they have been found to play a role as either an oncogene, tumor suppressor, or can mediate either of these effects in a context-dependent manner. Among the extensively studied CRL3-based E3 ligases, the role of the adaptor protein SPOP (speckle type BTB/POZ protein) in tumorigenesis appears to be tissue or cellular context dependent. Specifically, SPOP acts as a tumor suppressor via destabilizing downstream oncoproteins in many malignancies, especially in prostate cancer. However, SPOP has largely an oncogenic role in kidney cancer. Keap1, another well-characterized CRL3 adaptor protein, likely serves as a tumor suppressor within diverse malignancies, mainly due to its specific turnover of its downstream oncogenic substrate, NRF2 (nuclear factor erythroid 2-related factor 2). In accordance with the physiological role the various CRL3 adaptors exhibit, several pharmacological agents have been developed to disrupt its E3 ligase activity, therefore blocking its potential oncogenic activity to mitigate tumorigenesis.
Assuntos
Carcinogênese/genética , Proteínas Culina/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Carcinogênese/metabolismo , Proteínas Culina/genética , Humanos , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genéticaRESUMO
The individuals carrying melanocortin-1 receptor (MC1R) variants, especially those associated with red hair color, fair skin, and poor tanning ability (RHC trait), are more prone to melanoma; however, the underlying mechanism is poorly defined. Here, we report that UVB exposure triggers phosphatase and tensin homolog (PTEN) interaction with wild-type (WT), but not RHC-associated MC1R variants, which protects PTEN from WWP2-mediated degradation, leading to AKT inactivation. Strikingly, the biological consequences of the failure of MC1R variants to suppress PI3K/AKT signaling are highly context dependent. In primary melanocytes, hyperactivation of PI3K/AKT signaling leads to premature senescence; in the presence of BRAF(V600E), MC1R deficiency-induced elevated PI3K/AKT signaling drives oncogenic transformation. These studies establish the MC1R-PTEN axis as a central regulator for melanocytes' response to UVB exposure and reveal the molecular basis underlying the association between MC1R variants and melanomagenesis.
Assuntos
Regulação da Expressão Gênica/efeitos da radiação , Melanócitos/metabolismo , Melanoma Experimental/patologia , PTEN Fosfo-Hidrolase/metabolismo , Receptor Tipo 1 de Melanocortina/metabolismo , Pigmentação da Pele/fisiologia , Raios Ultravioleta , Animais , Western Blotting , Células Cultivadas , Humanos , Técnicas Imunoenzimáticas , Melanócitos/efeitos da radiação , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Mutação/genética , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Melanocortina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Pigmentação da Pele/efeitos da radiação , alfa-MSH/genética , alfa-MSH/metabolismoRESUMO
Proteasome-mediated degradation is a common mechanism by which cells renew their intracellular proteins and maintain protein homeostasis. In this process, the E3 ubiquitin ligases are responsible for targeting specific substrates (proteins) for ubiquitin-mediated degradation. However, in cancer cells, the stability and the balance between oncoproteins and tumor suppressor proteins are disturbed in part due to deregulated proteasome-mediated degradation. This ultimately leads to either stabilization of oncoprotein(s) or increased degradation of tumor suppressor(s), contributing to tumorigenesis and cancer progression. Therefore, E3 ubiquitin ligases including the SCF types of ubiquitin ligases have recently evolved as promising therapeutic targets for the development of novel anti-cancer drugs. In this review, we highlighted the critical components along the ubiquitin pathway including E1, E2, various E3 enzymes and DUBs that could serve as potential drug targets and also described the available bioactive compounds that target the ubiquitin pathway to control various cancers.
Assuntos
Terapia de Alvo Molecular/métodos , Neoplasias/terapia , Ubiquitina/metabolismo , Animais , Humanos , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/uso terapêutico , Transdução de Sinais , Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/fisiologia , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/fisiologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/fisiologia , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/fisiologia , Ubiquitinação/fisiologiaRESUMO
The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that primarily governs cell cycle progression. APC/C is composed of at least 14 core subunits and recruits its substrates for ubiquitination via one of the two adaptor proteins, Cdc20 or Cdh1, in M or M/early G1 phase, respectively. Furthermore, recent studies have shed light on crucial functions for APC/C in maintaining genomic integrity, neuronal differentiation, cellular metabolism and tumorigenesis. To gain better insight into the in vivo physiological functions of APC/C in regulating various cellular processes, particularly development and tumorigenesis, a number of mouse models of APC/C core subunits, coactivators or inhibitors have been established and characterized. However, due to their essential role in cell cycle regulation, most of the germline knockout mice targeting the APC/C pathway are embryonic lethal, indicating the need for generating conditional knockout mouse models to assess the role in tumorigenesis for each APC/C signaling component in specific tissues. In this review, we will first provide a brief introduction of the ubiquitin-proteasome system (UPS) and the biochemical activities and cellular functions of the APC/C E3 ligase. We will then focus primarily on characterizing genetic mouse models used to understand the physiological roles of each APC/C signaling component in embryogenesis, cell proliferation, development and carcinogenesis. Finally, we discuss future research directions to further elucidate the physiological contributions of APC/C components during tumorigenesis and validate their potentials as a novel class of anti-cancer targets.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Carcinogênese/genética , Neoplasias/genética , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Animais , Transformação Celular Neoplásica/genética , Humanos , Camundongos , Mitose , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genéticaRESUMO
Preimplantation development culminates with the emergence of three distinct populations: the inner cell mass, primitive endoderm and trophectoderm. Here, we define the mechanisms underlying the requirement of Suds3 in pre/peri-implantation development. Suds3 knockdown blastocysts exhibit a failure of both trophectoderm proliferation as well as a conspicuous lack of primitive endoderm. Expression of essential lineage factors Nanog, Sox2, Cdx2, Eomes, Elf5 and Sox17 are severely reduced in the absence of Suds3. Importantly, we document deficient FGF4/ERK signaling and show that exogenous FGF4 rescues primitive endoderm formation and trophectoderm proliferation in Suds3 knockdown blastocysts. We also show that Hdac1 knockdown reduces Sox2/FGF4/ERK signaling in blastocysts. Collectively, these data define a role for Suds3 in activation of FGF4/ERK signaling and determine an essential molecular role of Suds3/Sin3/HDAC complexes in lineage specification in vivo.
Assuntos
Padronização Corporal , Linhagem da Célula , Proteínas Repressoras/deficiência , Animais , Massa Celular Interna do Blastocisto/citologia , Massa Celular Interna do Blastocisto/efeitos dos fármacos , Massa Celular Interna do Blastocisto/metabolismo , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Ectoderma/citologia , Ectoderma/efeitos dos fármacos , Ectoderma/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fator 4 de Crescimento de Fibroblastos/metabolismo , Fator 4 de Crescimento de Fibroblastos/farmacologia , Fator de Transcrição GATA6/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Histona Desacetilase 1/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Proteína Homeobox Nanog , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXF/genética , Fatores de Transcrição SOXF/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
RNA transcription, processing and translation are fundamental molecular processes required for development, growth and cell viability. Towards the functional annotation of the genome, we are engaged in a reverse genetic screen using mammalian preimplantation embryos as a model system. Here we report the essential function of four RNA processing/splicing factors (Sf3b14, Sf3b1, Rpl7l1, and Rrp7a) and show that each of these genes is required for blastocyst formation in the mouse. As very little information is known about these genes, we characterized their normal expression and localization in mouse embryos as well as phenotypic analysis of loss of function during preimplantation development. Functional knockdown of each gene product results in normal morula development but there is failure to form a blastocoel cavity or morphologically differentiated trophectoderm. We show that zygotic genome activation does occur as well as initial lineage specification in the absence of each factor. Consistent with a role in RNA splicing, we demonstrate that the absence of Sf3b14 and Sf3b1 in 8-cell and morula-stage embryos results in a specific reduction of intron containing transcripts, but no reduction single-exon genes. Taken together, we show critical developmental and molecular requirements of Sf3b14, Sf3b1, Rpl7l1, and Rrp7a during mammalian preimplantation.
Assuntos
Blastocisto/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Proteínas Ribossômicas/genética , Animais , Feminino , Técnicas de Silenciamento de Genes , Masculino , Camundongos Endogâmicos , Mórula/fisiologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Splicing de RNA , Fatores de Processamento de RNARESUMO
Chimeric antigen receptor (CAR-T) cell therapy is a newly developed immunotherapy strategy and has achieved satisfactory outcomes in the treatment of hematological malignancies. However, some adverse effects related to CAR-T cell therapy have to be resolved before it is widely used in clinics as a cancer treatment. Furthermore, the application of CAR-T cell therapy in the treatment of solid tumors has been hampered by numerous limitations. Therefore, it is essential to explore novel strategies to improve the therapeutic effect of CAR-T cell therapy. In this review, we summarized the recently developed strategies aimed at optimizing the generation of CAR-T cells and improving the anti-tumor efficiency of CAR-T cell therapy. Furthermore, the discovery of new targets for CAR-T cell therapy and the combined treatment strategies of CAR-T cell therapy with chemotherapy, radiotherapy, cancer vaccines and nanomaterials are highlighted.
Assuntos
Imunoterapia Adotiva , Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Neoplasias/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Imunoterapia/métodos , Linfócitos T/imunologia , Terapia Combinada/métodos , Vacinas Anticâncer/uso terapêutico , Vacinas Anticâncer/imunologiaRESUMO
BACKGROUND: The number of patients with type 1 diabetes rises rapidly around the world in recent years. Maternal diabetes has a detrimental effect on reproductive outcomes due to decreased oocyte quality. However, the strategies to improve the oocyte quality and artificial reproductive technology (ART) efficiency of infertile females suffering from diabetes have not been fully studied. In this study, we aimed to examine the effects of nicotinamide mononucleotide (NMN) on oocyte maturation of mouse with type 1 diabetes mouse and explore the underlying mechanisms of NMN's effect. METHODS: Streptozotocin (STZ) was used to establish the mouse models with type 1 diabetes. The successful establishment of the models was confirmed by the results of body weight test, fasting blood glucose test and haematoxylin and eosin (H&E) staining. The in vitro maturation (IVM) rate of oocytes from diabetic mice was examined. Immunofluorescence staining (IF) was performed to examine the reactive oxygen species (ROS) level, spindle/chromosome structure, mitochondrial function, actin dynamics, DNA damage and histone modification of oocytes, which are potential factors affecting the oocyte quality. The quantitative reverse transcription PCR (RT-qPCR) was used to detect the mRNA levels of Sod1, Opa1, Mfn2, Drp1, Sirt1 and Sirt3 in oocytes. RESULTS: The NMN supplementation increased the oocyte maturation rate of the mice with diabetes. Furthermore, NMN supplementation improved the oocyte quality by rescuing the actin dynamics, reversing meiotic defects, improving the mitochondrial function, reducing ROS level, suppressing DNA damage and restoring changes in histone modifications of oocytes collected from the mice with diabetes. CONCLUSION: NMN could improve the maturation rate and quality of oocytes in STZ-induced diabetic mice, which provides a significant clue for the treatment of infertility of the patients with diabetes.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Dinaminas , Mononucleotídeo de Nicotinamida , Oócitos , Espécies Reativas de Oxigênio , Animais , Camundongos , Feminino , Oócitos/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Mononucleotídeo de Nicotinamida/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Sirtuína 1/metabolismo , Sirtuína 3/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Superóxido Dismutase-1 , Dano ao DNA/efeitos dos fármacos , Estreptozocina , Oogênese/efeitos dos fármacosRESUMO
It was well known that SPOP is highly mutated in various cancers especially the prostate cancer and SPOP mutation dramatically impaired its tumor suppressive function. However, the detailed role and underlying mechanisms of SPOP in regulating the growth of gastric cancer is not fully studied. Here, we found that Cullin3SPOP promoted the ubiquitination and degradation of TIAM1 protein in gastric cancer setting. Gastric cancer and prostate cancer derived SPOP mutation failed to suppress the proliferation, migration and invasion of gastric cancer cells partially due to the elevated level of TIAM1 protein. Notably, SPOP protein were negatively associated with TIAM1 protein in human gastric cancer tissue specimens. In conclusion, our results elucidate a molecular mechanism by which SPOP regulates the stability of TIAM1, and further demonstrate that SPOP inhibits the progression of gastric cancer by promoting the ubiquitination and degradation of TIAM1 protein.
Assuntos
Neoplasias da Próstata , Neoplasias Gástricas , Masculino , Humanos , Neoplasias Gástricas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/genética , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/patologia , UbiquitinaçãoRESUMO
Several research groups have suggested that the embryonic-abembryonic (Em-Ab) axis in the mouse can be predicted by the first cleavage plane of the early embryo. Currently, it is not known whether this early patterning occurs in cloned embryos produced by nuclear transfer and whether it affects development to term. In this work, the relationship between the first cleavage plane and the Em-Ab axis was determined by the labeling of one blastomere in cloned mouse embryos at the 2-cell stage, followed by ex-vivo tracking until the blastocyst stage. The results demonstrate that approximately half of the cloned blastocysts had an Em-Ab axis perpendicular to the initial cleavage plane of the 2-cell stage. These embryos were classified as "orthogonal" and the remainder as "deviant". Additionally, we report here that cloned embryos were significantly more often orthogonal than their naturally fertilized counterparts and overexpressed Sox2. Orthogonal cloned embryos demonstrated a higher rate of post-implantation embryonic development than deviant embryos, but cloned pups did not all survive. These results reveal that the angular relationship between the Em-Ab axis and the first cleavage plane can influence later development and they support the hypothesis that proper early patterning of mammalian embryos is required after nuclear transfer.
Assuntos
Blastocisto/citologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Animais , Blastocisto/metabolismo , Clonagem de Organismos , Transferência Embrionária , Embrião de Mamíferos/metabolismo , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microscopia Confocal , Proteína Homeobox Nanog , Técnicas de Transferência Nuclear , Fator 3 de Transcrição de Octâmero/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genéticaRESUMO
The mutation of the crucial genes such as tumor suppressors or oncogenes plays an important role in the initiation and development of tumors. The non-synonymous mutations in the tumor cell genome will produce non-autologous proteins (neoantigen) to activate the immune system by activating CD4+ and CD8+ T cells. Neoantigen-based peptide vaccines have exhibited exciting therapeutic effects in treating various cancers alone or in combination with other therapeutic strategies. Furthermore, antigen-loaded DC vaccines are more powerful in inducing stronger immune responses than vaccines generated by antigens and adjuvants. Therefore, neoantigen-based dendritic cell (DC) vaccines could achieve promising effects in combating some malignant tumors. In this review, we summarized and discussed the recent research progresses of the neoantigen, neoantigen-based vaccines, and DC-based vaccine in pancreatic cancers (PCs). The combination of the neoantigen and DC-based vaccine in PC was also highlighted. Therefore, our work will provide more detailed evidence and novel opinions to promote the development of a personalized neoantigen-based DC vaccine for PC.