RESUMO
Deficiency in the E3 ubiquitin ligase UBE3A leads to the neurodevelopmental disorder Angelman syndrome (AS), while additional dosage of UBE3A is linked to autism spectrum disorder. The mechanisms underlying the downstream effects of UBE3A gain or loss of function in these neurodevelopmental disorders are still not well understood, and effective treatments are lacking. Here, using stable-isotope labeling of amino acids in mammals and ubiquitination assays, we identify PTPA, an activator of protein phosphatase 2A (PP2A), as a bona fide ubiquitin ligase substrate of UBE3A. Maternal loss of Ube3a (Ube3am-/p+) increased PTPA level, promoted PP2A holoenzyme assembly, and elevated PP2A activity, while maternal 15q11-13 duplication containing Ube3a down-regulated PTPA level and lowered PP2A activity. Reducing PTPA level in vivo restored the defects in dendritic spine maturation in Ube3am-/p+ mice. Moreover, pharmacological inhibition of PP2A activity with the small molecule LB-100 alleviated both reduction in excitatory synaptic transmission and motor impairment in Ube3am-/p+ mice. Together, our results implicate a critical role of UBE3A-PTPA-PP2A signaling in the pathogenesis of UBE3A-related disorders and suggest that PP2A-based drugs could be potential therapeutic candidates for treatment of UBE3A-related disorders.
Assuntos
Espinhas Dendríticas/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteína Fosfatase 2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Transtorno do Espectro Autista/metabolismo , Encéfalo/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Camundongos , Camundongos Transgênicos , Proteína Fosfatase 2/antagonistas & inibidores , Proteólise , Transmissão Sináptica , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Malignant glioma is the most common and aggressive form of brain tumor with poor prognosis of survival. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent but is insufficient of inducing apoptosis in some types of gliomas. In this study, we showed that the small-molecule Mcl-1 inhibitor UMI-77 sensitized glioma cells to TRAIL treatment, as evidenced by cell viability assay, Annexin V staining and JC-1 staining. Combination of UMI-77 and TRAIL in glioma cells led to the activation of caspase-8 and Bid, cleavage of caspase-3 and poly-ADP ribose polymerase (PARP), accumulation of tBid in the mitochondria and release of cytochrome c into the cytosol. UMI-77 alone or in combination with TRAIL untethered pro-apoptotic Bcl-2 proteins Bim and Bak from the sequestration of Mcl-1 and promoted the conformational activation of Bak. Small hairpin RNA (shRNA) of Bid attenuated the cleavage of caspase-8, Bid, caspase-3 and PARP, and reduced the cytotoxicity of UMI-77 plus TRAIL as compared with control shRNA cells, indicating this synergy entails the crosstalk between extrinsic and intrinsic apoptotic signaling. Taken together, UMI-77 enhances TRAIL-induced apoptosis by unsequestering Bim and Bak, which provides a novel therapeutic strategy for the treatment of gliomas.
Assuntos
Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/metabolismo , Glioma/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Sulfonamidas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Tioglicolatos/farmacologia , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Glioma/tratamento farmacológico , Glioma/patologia , Células HeLa , HumanosRESUMO
XPO1 (exportin1) mediates nuclear export of proteins and RNAs and is frequently overexpressed in cancers. In this study, we show that the orally bioavailable XPO1 inhibitor KPT-330 reduced Mcl-1 protein level, by which it synergized with Bcl-xL inhibitor A-1331852 to induce apoptosis in cancer cells. KPT-330/A-1331852 combination disrupted bindings of Mcl-1 and Bcl-xL to Bax, Bak, and/or Bim, elicited mitochondrial outer membrane permeabilization, and triggered apoptosis. KPT-330 generally mitigated mRNA expression and protein synthesis rather than mRNA nuclear export or protein stability of Mcl-1. KPT-330 inhibited mTORC1/4E-BP1 and Mnk1/eIF4E axes, which disrupted the eIF4F translation initiation complex but was dispensable for Mcl-1 reduction and KPT-330/A-1331852 combination-induced apoptosis. Mature rRNAs are integral components of the ribosome that determines protein synthesis ability. KPT-330 impeded nucleolar rRNA processing and reduced total levels of multiple mature rRNAs. Reconstitution of XPO1 by expressing degradation-resistant C528S mutant retained rRNA amount, Mcl-1 expression, and Bcl-xL inhibitor resistance upon KPT-330 treatment. KPT-330/A-1331852 combination suppressed growth and enhanced apoptosis of non-small cell lung cancer xenografts. Therefore, we clarify the reason of apoptosis resistance of cancer cells to XPO1 inhibition and develop a potential strategy for treating solid tumors.
Assuntos
Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Hidrazinas/farmacologia , Isoquinolinas/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , RNA Ribossômico/metabolismo , Triazóis/farmacologia , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Benzotiazóis/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Fator de Iniciação 4F em Eucariotos/metabolismo , Humanos , Hidrazinas/uso terapêutico , Isoquinolinas/uso terapêutico , Carioferinas/antagonistas & inibidores , Carioferinas/genética , Carioferinas/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Triazóis/uso terapêutico , Proteína Exportina 1RESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with potential anticancer effect, but innate and adaptive TRAIL resistance in majority of cancers limit its clinical application. Karyopherin ß1 (KPNB1) inhibition in cancer cells has been reported to abrogate the nuclear import of TRAIL receptor DR5 and facilitate its localization on the cell surface ready for TRAIL stimulation. However, our study reveals a more complicated mechanism. Genetic or pharmacological inhibition of KPNB1 potentiated TRAIL-induced apoptosis selectively in glioblastoma cells mainly by unfolded protein response (UPR). First, it augmented ATF4-mediated DR5 expression and promoted the assembly of death-inducing signaling complex (DISC). Second, it freed Bax and Bak from Mcl-1. Third, it downregulated FLIPL and FLIPS, inhibitors of caspase-8 cleavage, partly through upregulating ATF4-induced 4E-BP1 expression and disrupting the cap-dependent translation initiation. Meanwhile, KPNB1 inhibition-induced undesirable autophagy and accelerated cleaved caspase-8 clearance. Inhibition of autophagic flux maintained cleaved caspase-8 and aggravated apoptosis induced by KPNB1 inhibitor plus TRAIL, which were abolished by caspase-8 inhibitor. These results unveil new molecular mechanism for optimizing TRAIL-directed therapeutic efficacy against cancer.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , beta Carioferinas/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Caspase 8/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/genéticaRESUMO
The nuclear import receptor karyopherin ß1 (KPNB1) is involved in the nuclear import of most proteins and in the regulation of multiple mitotic events. Upregulation of KPNB1 has been observed in cancers including glioblastoma. Depletion of KPNB1 induces mitotic arrest and apoptosis in cancer cells, but the underlying mechanism is not clearly elucidated. Here, we found that downregulation and functional inhibition of KPNB1 in glioblastoma cells induced growth arrest and apoptosis without apparent mitotic arrest. KPNB1 inhibition upregulated Puma and Noxa and freed Mcl-1-sequestered Bax and Bak, leading to mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Moreover, combination of Bcl-xL inhibitors and KPNB1 inhibition enhanced apoptosis in glioblastoma cells. KPNB1 inhibition promoted cytosolic retention of its cargo and impaired cellular proteostasis, resulting in elevated polyubiquitination, formation of aggresome-like-induced structure (ALIS), and unfolded protein response (UPR). Ubiquitination elevation and UPR activation in KPNB1-deficient cells were reversed by KPNB1 overexpression or inhibitors of protein synthesis but aggravated by inhibitors of autophagy-lysosome or proteasome, indicating that rebalance of cytosolic/nuclear protein distribution and alleviation of protein overload favor proteostasis and cell survival. Chronic activation of eIF2α/ATF4 cascade of UPR was responsible for the upregulation of Puma and Noxa, apoptosis and ABT-263 sensitivity. Taken together, our findings demonstrate that KPNB1 is required for proteostasis maintenance and its inhibition induces apoptosis in glioblastoma cells through UPR-mediated deregulation of Bcl-2 family members.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Resposta a Proteínas não Dobradas , beta Carioferinas/metabolismo , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Proteostase , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , UbiquitinaçãoRESUMO
Globin family was famous for oxygen supply function of its members such as hemoglobin and myoglobin. With the progress of research, several members of this protein family have been proven to play roles in tumors including glioma. Androglobin (ADGB) is a recently identified member of globin family with very few studies about its function. In the present study, we show that ADGB plays an oncogene role in glioma. Lentiviral vector mediated ADGB knockdown inhibited the proliferation of glioma cell lines determined by MTT assay and colony formation assay. ADGB knockdown also increased the apoptosis of glioma cell line U251 assessed by flow cytometry. In addition, western blot showed that ADGB knockdown altered levels of several proteins related to proliferation, survival or apoptosis in U251 cells. These findings suggest ADGB is involved in the progression of glioma in vitro.
Assuntos
Neoplasias Encefálicas/metabolismo , Proteínas de Ligação a Calmodulina/metabolismo , Proliferação de Células , Glioma/metabolismo , Globinas/metabolismo , Apoptose , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Vetores Genéticos , Glioma/genética , Glioma/patologia , Globinas/genética , Humanos , Lentivirus/genética , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
DSTYK (Dual serine/threonine and tyrosine protein kinase) is a putative dual Ser/Thr and Tyr protein kinase with unique structural features. It is proposed that DSTYK may play important roles in brain because of its high expression in most brain areas. In the present study, a DSTYK knockout (KO) mouse line with the ablation of C-terminal of DSTYK including the kinase domain was generated to study the physiological function of DSTYK. The DSTYK KO mice are fertile and have no significant morphological defects revealed by Nissl staining compared with wildtype mice. Open field test and rotarod test showed there is no obvious difference in basic motor and balance capacity between the DSTYK homozygous KO mice and DSTYK heterozygous KO mice. In water maze test, however, the DSTYK homozygous KO mice show impaired capabilities of learning and memory compared with the DSTYK heterozygous KO mice.
Assuntos
Comportamento Animal , Deficiências da Aprendizagem/enzimologia , Aprendizagem em Labirinto , Transtornos da Memória/enzimologia , Memória , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Animais , Genótipo , Deficiências da Aprendizagem/genética , Deficiências da Aprendizagem/psicologia , Masculino , Transtornos da Memória/genética , Transtornos da Memória/psicologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Fenótipo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Teste de Desempenho do Rota-RodRESUMO
Arcuate nucleus of hypothalamus (ARH) is the core component in the regulation circuits of food intake and energy homeostasis. ARH projections to other parts of the hypothalamus and to extrahypothalamic areas are established in the postnatal two weeks, which is a pivotal stage for individual development. ß-Catenin, a cell adhesion protein and also the mediator of canonical Wnt signaling pathway, plays an important role in embryonic development and adult homeostasis. However, whether ß-catenin plays any roles in the development of hypothalamus is not clear. Here, we report that perinatal conditional knockout of ß-catenin by CamKIIα-Cre in forebrain reduces body weight gain from P8 and dramatically shortens life span. Quantitative PCR and in situ hybridization results showed the expression of NPY mRNA in the ARH of ß-catenin CKO mice at P15 is obviously increased compared with that of littermate controls, whereas the expression of POMC mRNA is significantly decreased, which suggested the reduction of postnatal body weight gain might be due to the deficiency of food intake. Together, ß-catenin might play an important role in the regulation of food intake and postnatal body weight gain probably through affecting the development of ARH circuits.
Assuntos
Núcleo Arqueado do Hipotálamo/fisiologia , Peso Corporal/genética , Ingestão de Alimentos/genética , Aumento de Peso/genética , beta Catenina/genética , Animais , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Neuropeptídeo Y/metabolismo , Pró-Opiomelanocortina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta Catenina/metabolismoRESUMO
ZFX (zinc finger transcription factor, X chromosome-linked) contributes to the maintenance of different types of stem cells and the progression of various cancers. We have previously reported that ZFX knockdown inhibits proliferation of glioma in vitro and in vivo. Since overexpression of ZFX in lung cancer tissue correlates with lymph node metastasis, we hypothesized that ZFX may play a role in lung cancer. In this study, we identified ZFX as a promoter of lung cancer growth and migration in a NSCLC (non-small cell lung carcinoma) cell line H1299. ZFX knockdown caused proliferation inhibition determined by MTT assay and colony formation assay, G0/G1 arrest of cell cycle and slightly increased proportion of apoptotic cells assessed by flow cytometry assay, decreased population of migrating cells showed by wound-healing assay, increased cell senescence evidenced by senescence-associated ß-galactosidase staining. ZFX knockdown also led to decreased proportion of tumor bearing mice and reduced mean tumor volume in a subcutaneous tumor model. In addition, western blot showed that ZFX knockdown down regulated a set of proteins involved in proliferation, survival and motility. Altogether, these results suggest that ZFX may be a potential therapeutic target for NSCLC.