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1.
Nat Commun ; 12(1): 4919, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389725

RESUMO

BRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of ß-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed ß-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, ß-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that ß-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Oncogenes/genética , Transcrição Genética/genética , beta Catenina/genética , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Feminino , Perfilação da Expressão Gênica/métodos , Células HeLa , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA-Seq/métodos , beta Catenina/metabolismo
2.
Nat Commun ; 12(1): 4928, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389720

RESUMO

Diabetes results from a decline in functional pancreatic ß-cells, but the molecular mechanisms underlying the pathological ß-cell failure are poorly understood. Here we report that large-tumor suppressor 2 (LATS2), a core component of the Hippo signaling pathway, is activated under diabetic conditions and induces ß-cell apoptosis and impaired function. LATS2 deficiency in ß-cells and primary isolated human islets as well as ß-cell specific LATS2 ablation in mice improves ß-cell viability, insulin secretion and ß-cell mass and ameliorates diabetes development. LATS2 activates mechanistic target of rapamycin complex 1 (mTORC1), a physiological suppressor of autophagy, in ß-cells and genetic and pharmacological inhibition of mTORC1 counteracts the pro-apoptotic action of activated LATS2. We further show a direct interplay between Hippo and autophagy, in which LATS2 is an autophagy substrate. On the other hand, LATS2 regulates ß-cell apoptosis triggered by impaired autophagy suggesting an existence of a stress-sensitive multicomponent cellular loop coordinating ß-cell compensation and survival. Our data reveal an important role for LATS2 in pancreatic ß-cell turnover and suggest LATS2 as a potential therapeutic target to improve pancreatic ß-cell survival and function in diabetes.


Assuntos
Autofagia , Diabetes Mellitus/metabolismo , Células Secretoras de Insulina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Células Cultivadas , Diabetes Mellitus/genética , Diabetes Mellitus/patologia , Humanos , Células Secretoras de Insulina/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Ratos , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/genética
3.
FASEB J ; 35(9): e21827, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383980

RESUMO

Neuron-derived orphan receptor 1, NR4A3 (Nor1)/NR4A3 is an orphan nuclear receptor involved in the transcriptional control of developmental and neurological functions. Oxidative stress-induced conditions are primarily associated with neurological defects in humans, yet the impact on Nor1-mediated transcription of neuronal genes remains with unknown mechanism. Here, we demonstrate that Nor1 is a non-conventional target of SUMO2/3 conjugation at Lys-137 contained in an atypic ψKxSP motif referred to as the pSuM. Nor1 pSuM SUMOylation differs from the canonical process with the obligate phosphorylation of Ser-139 by Ras signaling to create the required negatively charged interface for SUMOylation. Additional phosphorylation at sites flanking the pSuM is also mediated by the coordinated action of protein kinase casein kinase 2 to function as a small ubiquitin-like modifier enhancer, regulating Nor1-mediated transcription and proteasomal degradation. Nor1 responsive genes involved in cell proliferation and metabolism, such as activating transcription factor 3, cyclin D1, CASP8 and FADD-like apoptosis regulator, and enolase 3 were upregulated in response to pSuM disruption in mouse HT-22 hippocampal neuronal cells and human neuroblastoma SH-SY5Y cells. We also identified critical antioxidant genes, such as catalase, superoxide dismutase 1, and microsomal glutathione S-transferase 2, as responsive targets of Nor1 under pSuM regulation. Nor1 SUMOylation impaired gene transcription through less effective Nor1 chromatin binding and reduced enrichment of histone H3K27ac marks to gene promoters. These effects resulted in decreased neuronal cell growth, increased apoptosis, and reduced survival to oxidative stress damage, underlying the role of pSuM-modified Nor1 in redox homeostasis. Our findings uncover a hierarchical post-translational mechanism that dictates Nor1 non-canonical SUMOylation, disrupting Nor1 transcriptional competence, and neuroprotective redox sensitivity.


Assuntos
Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Sumoilação/genética , Animais , Apoptose/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Quinase do Ponto de Checagem 2/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Hipocampo/metabolismo , Homeostase/genética , Humanos , Camundongos , Neuroblastoma/genética , Neurônios/metabolismo , Oxirredução , Estresse Oxidativo/genética , Fosforilação/genética , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Regulação para Cima/genética
4.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360757

RESUMO

Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the pre-mir-146b structure to target miR-146b-5p and miR-146b-3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-miR-146b-GuideA-puromycin and pSp-Cas9n-miR-146b-GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes , MicroRNAs , RNA Neoplásico , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Animais , Linhagem Celular , Movimento Celular/genética , Sobrevivência Celular/genética , Xenoenxertos , Humanos , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , Transplante de Neoplasias , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia
5.
Cancer Sci ; 112(9): 3585-3597, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34252986

RESUMO

Diffuse large B cell lymphoma (DLBCL) heterogeneity promotes recurrence and anti-CD20-based therapeutic resistance. Previous studies have shown that downregulation of MS4A1/CD20 expression after chemoimmunotherapy with rituximab leads to rituximab resistance. However, the mechanisms of CD20 loss remain unknown. We identified that pyruvate dehydrogenase kinase 4 (PDK4) is markedly elevated in DLBCL cells derived from both patients and cell lines with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) resistance. We found that overexpression of PDK4 in DLBCL cells resulted in cell proliferation and resistance to rituximab in vitro and in vivo. Furthermore, loss of PDK4 expression or treatment with the PDK4 inhibitor dichloroacetate was able to significantly increase rituximab-induced cell apoptosis in DLBCL cells. Further studies suggested PDK4 mediates a metabolic shift, in that the main energy source was changed from oxidative phosphorylation to glycolysis, and the metabolic changes could play an important role in rituximab resistance. Importantly, by knocking down or overexpressing PDK4 in DLBCL cells, we showed that PDK4 has a negative regulation effect on MS4A1/CD20 expression. Collectively, this is the first study showing that targeting PDK4 has the potential to overcome rituximab resistance in DLBCL.


Assuntos
Antígenos CD20/metabolismo , Antineoplásicos Imunológicos/administração & dosagem , Reprogramação Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Glicoproteínas/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Rituximab/administração & dosagem , Transdução de Sinais/genética , Adolescente , Adulto , Idoso , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Transfecção , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
6.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203429

RESUMO

DDX3 RNA helicase is intensively studied as a therapeutic target due to participation in the replication of some viruses and involvement in cancer progression. Here we used transcriptome analysis to estimate the primary response of hepatocytes to different levels of RNAi-mediated knockdown of DDX3 RNA helicase both in vitro and in vivo. We found that a strong reduction of DDX3 protein (>85%) led to similar changes in vitro and in vivo-deregulation of the cell cycle and Wnt and cadherin pathways. Also, we observed the appearance of dead hepatocytes in the healthy liver and a decrease of cell viability in vitro after prolonged treatment. However, more modest downregulation of the DDX3 protein (60-65%) showed discordant results in vitro and in vivo-similar changes in vitro as in the case of strong knockdown and a different phenotype in vivo. These results demonstrate that the level of DDX3 protein can dramatically influence the cell phenotype in vivo and the decrease of DDX3, for more than 85% leads to cell death in normal tissues, which should be taken into account during the drug development of DDX3 inhibitors.


Assuntos
RNA Helicases DEAD-box/metabolismo , Hepatócitos/metabolismo , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , RNA Helicases DEAD-box/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Transcriptoma/genética
7.
Commun Biol ; 4(1): 834, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215850

RESUMO

The multiplexed cancer cell line screening platform PRISM demonstrated its utility in testing hundreds of cell lines in a single run, possessing the potential to speed up anti-cancer drug discovery, validation and optimization. Here we described the development and implementation of a next-generation PRISM platform combining Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing, cell line DNA barcoding and next-generation sequencing to enable genetic and/or pharmacological assessment of target addiction in hundreds of cell lines simultaneously. Both compound and CRISPR-knockout PRISM screens well recapitulated the results from individual assays and showed high consistency with a public database.


Assuntos
Antineoplásicos/farmacologia , Sistemas CRISPR-Cas , Detecção Precoce de Câncer/métodos , Edição de Genes/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células HEK293 , Humanos , Neoplasias/diagnóstico
8.
Anticancer Res ; 41(8): 3731-3740, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281831

RESUMO

BACKGROUND: The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is thought to have promising clinical potential. However, the off-target effects of Cas9 are a major concern for its application. Therefore, we hypothesized that the adverse effects of off-target gene editing might be minimized if the human codon-optimized Streptococcus pyogenes Cas9 (hCas9) could be specifically expressed in cancer cells. MATERIALS AND METHODS: We constructed a chimeric adenoviral vector, Ad5F35-MKp-hCas9, and infected human bladder cancer cell lines with this vector. The confirmation of hCas9 gene expression was performed in 3-4 days after from infection. RESULTS: hCas9 gene expression was observed in Ad5F35-MKp-hCas9 infected bladder cancer cells but not in non-malignant cells. CONCLUSION: Our study showed that the Ad5F35-MKp-hCas9 vector is capable of expressing the hCas9 gene with high specificity in bladder cancer cells. These findings may help in minimizing the risk of off-target effects of gene editing.


Assuntos
Adenoviridae/genética , Proteína 9 Associada à CRISPR/genética , Vetores Genéticos/genética , Transfecção/métodos , Neoplasias da Bexiga Urinária/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Edição de Genes/métodos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Regiões Promotoras Genéticas , Neoplasias da Bexiga Urinária/patologia
9.
DNA Cell Biol ; 40(7): 979-987, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34227845

RESUMO

Long noncoding RNA X-inactive specific transcript (XIST) has been identified as a crucial regulator in neurodegenerative disorders. However, the role and mechanism of XIST in ischemic stroke remain elusive. In our study, we found that XIST expression was upregulated in both mice subjected to middle cerebral artery occlusion and oxygen-glucose deprivation (OGD)-treated neurons. Functional assays disclosed that the interference of XIST accelerated viability, and suppressed apoptosis and caspase-3 activity in OGD-treated neurons. Moreover, XIST interacted with miR-98, and miR-98 targeted BTB-to-CNC homology 1 (BACH1). miR-98 silencing or BACH1 overexpression counteracted XIST knockdown-mediated effects on cell viability and apoptosis in OGD-treated neurons. In conclusion, our data demonstrated that XIST facilitated the progression of ischemic stroke through regulating the miR-98/BACH1 axis. These findings might provide a novel therapeutic strategy for ischemic stroke treatment.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , AVC Isquêmico/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Animais , Apoptose/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Isquemia Encefálica/genética , Sobrevivência Celular/genética , China , Glucose/metabolismo , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/metabolismo , AVC Isquêmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Neurônios/metabolismo , Oxigênio/metabolismo , RNA Longo não Codificante/metabolismo , Acidente Vascular Cerebral/genética
10.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203249

RESUMO

By providing ~70% of the eye's refractive power, the preocular tear film is essential for optimal vision. However, its integrity is often jeopardized by environmental and pathologic conditions that accelerate evaporation and cause sight-impairing dry eye. A key adaptive response to evaporation-induced tear film hyperosmolarity is the reflex-triggered release of tear-stabilizing mucin from conjunctival goblet cells. Here, we review progress in elucidating the roles of ion channels in mediating this important exocytotic response. Much is now known about the modulatory impact of ATP-sensitive potassium channels, nonspecific cation channels and voltage-gated calcium channels. Recently, we discovered that during unremitting extracellular hyperosmolarity, P2X7 receptor/channels also become activated and markedly impair goblet cell viability. However, our understanding of possible adaptive benefits of this P2X7 activation remains limited. In the present study, we utilized high-temporal resolution membrane capacitance measurements to monitor the exocytotic activity of single goblet cells located in freshly excised rat conjunctiva. We now report that activation of P2X7 purinoceptors boosts neural-evoked exocytosis and accelerates replenishment of mucin-filled granules after exocytotic depletion. Thus, P2X7 activation exerts a yin-yang effect on conjunctival goblet cells: the high-gain benefit of enhancing the supply of tear-stabilizing mucin is implemented at the high-risk of endangering goblet cell survival.


Assuntos
Células Caliciformes/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X/metabolismo , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Exocitose/genética , Exocitose/fisiologia , Humanos , Receptores Purinérgicos P2X/genética
11.
Nat Commun ; 12(1): 4371, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272364

RESUMO

Metabolic programming and mitochondrial dynamics along with T cell differentiation affect T cell fate and memory development; however, how to control metabolic reprogramming and mitochondrial dynamics in T cell memory development is unclear. Here, we provide evidence that the SUMO protease SENP1 promotes T cell memory development via Sirt3 deSUMOylation. SENP1-Sirt3 signalling augments the deacetylase activity of Sirt3, promoting both OXPHOS and mitochondrial fusion. Mechanistically, SENP1 activates Sirt3 deacetylase activity in T cell mitochondria, leading to reduction of the acetylation of mitochondrial metalloprotease YME1L1. Consequently, deacetylation of YME1L1 suppresses its activity on OPA1 cleavage to facilitate mitochondrial fusion, which results in T cell survival and promotes T cell memory development. We also show that the glycolytic intermediate fructose-1,6-bisphosphate (FBP) as a negative regulator suppresses AMPK-mediated activation of the SENP1-Sirt3 axis and reduces memory development. Moreover, glucose limitation reduces FBP production and activates AMPK during T cell memory development. These data show that glucose limitation activates AMPK and the subsequent SENP1-Sirt3 signalling for T cell memory development.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Linfócitos T CD8-Positivos/imunologia , Cisteína Endopeptidases/metabolismo , Memória Imunológica , Mitocôndrias/metabolismo , Sirtuína 3/metabolismo , Linfócitos T/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Acetilação , Aloenxertos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Neoplasias do Colo/imunologia , Frutosedifosfatos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Glucose/deficiência , Memória Imunológica/genética , Metabolômica , Metaloendopeptidases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Sirtuína 3/antagonistas & inibidores , Sirtuína 3/genética , Sumoilação , Linfócitos T/imunologia
12.
Nat Commun ; 12(1): 4375, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272366

RESUMO

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Piridazinas/química , Monofosfato de Adenosina/química , Varredura Diferencial de Calorimetria , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Microscopia Crioeletrônica , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Endorribonucleases/química , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Espectrometria de Massas , Complexos Multienzimáticos/ultraestrutura , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Piridazinas/farmacologia , Proteínas Recombinantes , Tetra-Hidroisoquinolinas/química
13.
Nat Commun ; 12(1): 4373, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272385

RESUMO

Although homologous recombination (HR) is indicated as a high-fidelity repair mechanism, break-induced replication (BIR), a subtype of HR, is a mutagenic mechanism that leads to chromosome rearrangements. It remains poorly understood how cells suppress mutagenic BIR. Trapping of Topoisomerase 1 by camptothecin (CPT) in a cleavage complex on the DNA can be transformed into single-ended double-strand breaks (seDSBs) upon DNA replication or colliding with transcriptional machinery. Here, we demonstrate a role of Abraxas in limiting seDSBs undergoing BIR-dependent mitotic DNA synthesis. Through counteracting K63-linked ubiquitin modification, Abraxas restricts SLX4/Mus81 recruitment to CPT damage sites for cleavage and subsequent resection processed by MRE11 endonuclease, CtIP, and DNA2/BLM. Uncontrolled SLX4/MUS81 loading and excessive end resection due to Abraxas-deficiency leads to increased mitotic DNA synthesis via RAD52- and POLD3- dependent, RAD51-independent BIR and extensive chromosome aberrations. Our work implicates Abraxas/BRCA1-A complex as a critical regulator that restrains BIR for protection of genome stability.


Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Recombinases/metabolismo , Animais , Camptotecina/farmacologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Recombinação Homóloga , Humanos , Proteína Homóloga a MRE11/metabolismo , Camundongos , RNA Interferente Pequeno , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Inibidores da Topoisomerase I/farmacologia , Ubiquitinação
14.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34201962

RESUMO

Sphingosine-1-phosphate (S1P) is a crucial mediator involved in the progression of different cancers, including glioblastoma multiforme (GBM), the most frequent and deadly human brain tumor, characterized by extensive invasiveness and rapid cell growth. Most of GBMs overexpress the epidermal growth factor receptor (EGFR), and we investigated the possible link between S1P and EGFR signaling pathways, focusing on its role in GBM survival, using the U87MG human cell line overexpressing EGFR (EGFR+). We previously demonstrated that EGFR+ cells have higher levels of extracellular S1P and increased sphingosine kinase-1 (SK1) activity than empty vector expressing cells. Notably, we demonstrated that EGFR+ cells are resistant to temozolomide (TMZ), the standard chemotherapeutic drug in GBM treatment, and the inhibition of SK1 or S1P receptors made EGFR+ cells sensitive to TMZ; moreover, exogenous S1P reverted this effect, thus involving extracellular S1P as a survival signal in TMZ resistance in GBM cells. In addition, both PI3K/AKT and MAPK inhibitors markedly reduced cell survival, suggesting that the enhanced resistance to TMZ of EGFR+ cells is dependent on the increased S1P secretion, downstream of the EGFR-ERK-SK1-S1P pathway. Altogether, our study provides evidence of a functional link between S1P and EGFR signaling pathways enhancing the survival properties of GBM cells.


Assuntos
Regulação Neoplásica da Expressão Gênica , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Antineoplásicos/farmacologia , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Humanos , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingosina/metabolismo
15.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299011

RESUMO

Osteoporosis is the most common metabolic bone disorder and nitrogen-containing bisphosphonates (BP) are a first line treatment for it. Yet, atypical femoral fractures (AFF), a rare adverse effect, may appear after prolonged BP administration. Given the low incidence of AFF, an underlying genetic cause that increases the susceptibility to these fractures is suspected. Previous studies uncovered rare CYP1A1 mutations in osteoporosis patients who suffered AFF after long-term BP treatment. CYP1A1 is involved in drug metabolism and steroid catabolism, making it an interesting candidate. However, a functional validation for the AFF-associated CYP1A1 mutations was lacking. Here we tested the enzymatic activity of four such CYP1A1 variants, by transfecting them into Saos-2 cells. We also tested the effect of commonly used BPs on the enzymatic activity of the CYP1A1 forms. We demonstrated that the p.Arg98Trp and p.Arg136His CYP1A1 variants have a significant negative effect on enzymatic activity. Moreover, all the BP treatments decreased CYP1A1 activity, although no specific interaction with CYP1A1 variants was found. Our results provide functional support to the hypothesis that an additive effect between CYP1A1 heterozygous mutations p.Arg98Trp and p.Arg136His, other rare mutations and long-term BP exposure might generate susceptibility to AFF.


Assuntos
Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Fraturas do Fêmur/genética , Fraturas do Fêmur/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Citocromo P-450 CYP1A1/química , Difosfonatos/uso terapêutico , Fraturas do Fêmur/enzimologia , Humanos , Incidência , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Filogenia , Alinhamento de Sequência
16.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299025

RESUMO

Several studies have demonstrated that melanoma-derived extracellular vesicles (EVs) are involved in lymph node metastasis; however, the molecular mechanisms involved are not completely defined. Here, we found that EMILIN-1 is proteolyzed and secreted in small EVs (sEVs) as a novel mechanism to reduce its intracellular levels favoring metastasis in mouse melanoma lymph node metastatic cells. Interestingly, we observed that EMILIN-1 has intrinsic tumor and metastasis suppressive-like properties reducing effective migration, cell viability, primary tumor growth, and metastasis. Overall, our analysis suggests that the inactivation of EMILIN-1 by proteolysis and secretion in sEVs reduce its intrinsic tumor suppressive activities in melanoma favoring tumor progression and metastasis.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Vesículas Extracelulares/metabolismo , Melanoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Biologia Computacional , Metástase Linfática/genética , Masculino , Espectrometria de Massas , Melanoma/genética , Melanoma/patologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não Paramétricas , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Cancer Sci ; 112(9): 3711-3721, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34107118

RESUMO

Antimitotic drugs such as vinca alkaloids and taxanes cause mitotic cell death after prolonged mitotic arrest. However, a fraction of cells escape from mitotic arrest by undergoing mitotic slippage, which is related to resistance to antimitotic drugs. Tipping the balance to mitotic cell death thus can be a way to overcome the drug resistance. Here we found that depletion of a mitotic regulator, CHAMP1 (chromosome alignment-maintaining phosphoprotein, CAMP), accelerates the timing of mitotic cell death after mitotic arrest. Live cell imaging revealed that CHAMP1-depleted cells died earlier than mock-treated cells in the presence of antimitotic drugs that resulted in the reduction of cells undergoing mitotic slippage. Depletion CHAMP1 reduces the expression of antiapoptotic Bcl-2 family proteins, especially Mcl-1. We found that CHAMP1 maintains Mcl-1 expression both at protein and mRNA levels independently of the cell cycle. At the protein level, CHAMP1 maintains Mcl-1 stability by suppressing proteasome-dependent degradation. Depletion of CHAMP1 reduces cell viability, and exhibits synergistic effects with antimitotic drugs. Our data suggest that CHAMP1 plays a role in the maintenance of Mcl-1 expression, implying that CHAMP1 can be a target to overcome the resistance to antimitotic drugs.


Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transdução de Sinais/genética , Células A549 , Animais , Antimitóticos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitose/efeitos dos fármacos , Mitose/genética , Neoplasias/genética , Neoplasias/patologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transfecção , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Cancer Sci ; 112(9): 3810-3821, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34145929

RESUMO

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in patients with non-small-cell lung cancer (NSCLC) harboring EGFR mutations. However, due to acquired resistance to EGFR-TKIs, even patients on third-generation osimertinib have a poor prognosis. Resistance mechanisms are still not fully understood. Here, we demonstrate that the increased expression of MUSASHI-2 (MSI2), an RNA-binding protein, is a novel mechanism for resistance to EGFR-TKIs. We found that after a long-term exposure to gefitinib, the first-generation EGFR-TKI lung cancer cells harboring the EGFR-TKI-sensitive mutations became resistant to both gefitinib and osimertinib. Although other mutations in EGFR were not found, expression levels of Nanog, a stemness core protein, and activities of aldehyde dehydrogenase (ALDH) were increased, suggesting that cancer stem-like properties were increased. Transcriptome analysis revealed that MSI2 was among the stemness-related genes highly upregulated in EGFR-TKI-resistant cells. Knockdown of MSI2 reduced cancer stem-like properties, including the expression levels of Nanog, a core stemness factor. We demonstrated that knockdown of MSI2 restored sensitivity to osimertinib or gefitinib in EGFR-TKI-resistant cells to levels similar to those of parental cells in vitro. An RNA immunoprecipitation (RIP) assay revealed that antibodies against MSI2 were bound to Nanog mRNA, suggesting that MSI2 increases Nanog expression by binding to Nanog mRNA. Moreover, overexpression of MSI2 or Nanog conferred resistance to osimertinib or gefitinib in parental cells. Finally, MSI2 knockdown greatly increased the sensitivity to osimertinib in vivo. Collectively, our findings provide proof of principle that targeting the MSI2-Nanog axis in combination with EGFR-TKIs would effectively prevent the emergence of acquired resistance.


Assuntos
Acrilamidas/farmacologia , Adenocarcinoma de Pulmão/metabolismo , Compostos de Anilina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Gefitinibe/farmacologia , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Ligação a RNA/metabolismo , Regulação para Cima , Células A549 , Acrilamidas/uso terapêutico , Adenocarcinoma de Pulmão/patologia , Compostos de Anilina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Gefitinibe/uso terapêutico , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/patologia , Mutação , Proteína Homeobox Nanog/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas de Ligação a RNA/genética , Transcriptoma , Transfecção
19.
Cancer Sci ; 112(9): 3784-3795, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34145930

RESUMO

Leptomeningeal carcinomatosis (LMC) occurs frequently in non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations and is associated with acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, the mechanism by which LMC acquires resistance to osimertinib, a third-generation EGFR-TKI, is unclear. In this study, we elucidated the resistance mechanism and searched for a novel therapeutic strategy. We induced osimertinib resistance in a mouse model of LMC using an EGFR-mutant NSCLC cell line (PC9) via continuous oral osimertinib treatment and administration of established resistant cells and examined the resistance mechanism using next-generation sequencing. We detected the Kirsten rat sarcoma (KRAS)-G12V mutation in resistant cells, which retained the EGFR exon 19 deletion. Experiments involving KRAS knockdown in resistant cells and KRAS-G12V overexpression in parental cells revealed the involvement of KRAS-G12V in osimertinib resistance. Cotreatment with trametinib (a MEK inhibitor) and osimertinib resensitized the cells to osimertinib. Furthermore, in the mouse model of LMC with resistant cells, combined osimertinib and trametinib treatment successfully controlled LMC progression. These findings suggest a potential novel therapy against KRAS-G12V-harboring osimertinib-resistant LMC in EGFR-mutant NSCLC.


Assuntos
Acrilamidas/administração & dosagem , Compostos de Anilina/administração & dosagem , Antineoplásicos/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Códon/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Carcinomatose Meníngea/tratamento farmacológico , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas p21(ras)/genética , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Carcinomatose Meníngea/genética , Carcinomatose Meníngea/metabolismo , Camundongos , Camundongos SCID , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Piridonas/farmacologia , Pirimidinonas/farmacologia , Transfecção , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Cancer Sci ; 112(9): 3545-3554, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34159680

RESUMO

The tumor microenvironment affects malignancy in hepatocellular carcinoma (HCC) cells, and cancer-associated fibroblasts (CAFs) play an important role in the microenvironment. As recent studies indicated a difference between CAFs isolated from chemoresistant and non-resistant cancer tissues, therefore we investigated the intracellular mechanism in resistant HCC co-cultured CAFs and interactions between these CAFs with cancer cells. We established a sorafenib-resistant (SR) Huh7 (human HCC) cell line, and characterized it with cytokine assays, then developed CAFs by co-culturing human hepatic stellate cells with resistant or parental Huh7 cells. The 2 types of CAFs were co-cultured with parental Huh7 cells, thereafter the cell viability of these Huh7 cells was checked under sorafenib treatment. The SR Huh7 (Huh7SR ) cells expressed increased B-cell activating factor (BAFF), which promoted high expression of CAF-specific markers in Huh7SR -co-cultured CAFs, showed activated BAFF, BAFF-R, and downstream of the NFκB-Nrf2 pathway, and aggravated invasion, migration, and drug resistance in co-cultured Huh7 cells. When we knocked down BAFF expression in Huh7SR cells, the previously increased malignancy and BAFF/NFκB axis in Huh7SR -co-cultured CAFs reversed, and enhanced chemoresistance in co-cultured Huh7 cells returned as well. In conclusion, the BAFF/NFκB pathway was activated in CAFs co-cultured with cell-culture medium from resistant Huh7, which promoted chemoresistance, and increased the malignancy in co-cultured non-resistant Huh7 cells. This suggests that the BAFF/NFκB axis in CAFs might be a potential therapeutic target in chemoresistance of HCC.


Assuntos
Antineoplásicos/farmacologia , Fator Ativador de Células B/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Carcinoma Hepatocelular/metabolismo , Comunicação Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Hepáticas/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/genética , Sorafenibe/farmacologia , Fator Ativador de Células B/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Transfecção
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