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INTRODUCTION: Homologous recombination repair during meiosis is essential for the exchange of genetic information between sister chromosomes, underpinning spermatogenesis and, consequently, fertility. The disruption of this process can lead to infertility, highlighting the importance of identifying the molecular actors involved. OBJECTIVES: This study aims to elucidate the role of the E3 ubiquitin ligase Rnf126 in spermatogenesis and its impact on fertility, particularly through its involvement in meiotic homologous recombination repair. METHODS: We used heterozygous and homozygous Rnf126 deletion models in mouse testes to examine the consequences on testicular health, sperm count, and the process of spermatogenesis. Additionally, we explored the association between RNF126 gene missense variants and nonobstructive male infertility in patients, with a focus on their functional impact on the protein's ubiquitin ligase activity. RESULTS: Rnf126 deletion led to testicular atrophy, disrupted seminiferous tubule structure, reduced sperm count, and spermatogenesis arrest at meiotic prophase I. Furthermore, male mice exhibited impaired homologous recombination repair and increased apoptosis within the seminiferous tubules. We identified four missense variants of the RNF126 gene (V68M, R241H, E261A, D253N) associated with male infertility. Specifically, the E261A and D253N variants, located in the RING domain, directly compromised the E3 ubiquitin ligase activity of RNF126. CONCLUSION: Our findings demonstrate the pivotal role of RNF126 in maintaining spermatogenesis and fertility, offering insights into the molecular mechanisms underlying male infertility. The identified RNF126 variants present novel targets for diagnostic and therapeutic strategies in treating nonobstructive male infertility.
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Although anti-HER2 therapy has made significant strides in reducing metastasis and relapse in HER2-positive breast cancer, resistance to agents like trastuzumab, pertuzumab, and lapatinib frequently develops in patients undergoing treatment. Previous studies suggest that the hyperactivation of the PI3K-AKT signaling pathway by PIK3CA/PTEN gene mutations is implicated in HER2 resistance. In this study, we introduce a novel PI3K-p110α Proteolysis TAargeting Chimera (PROTAC) that effectively inhibits the proliferation of breast cancer cells by degrading PI3K-p110α. When tested in two lapatinib-resistant cell lines, JIMT1 and MDA-MB-453, both of which harbor PIK3CA mutations, the PI3K PROTAC notably reduced cell proliferation and induced G1 phase cell cycle arrest. Importantly, even at very low concentrations, PI3K PROTAC restored sensitivity to lapatinib. Furthermore, the efficacy of PI3K PROTAC surpassed that of Alpelisib, a selective PI3K-p110α kinase inhibitor in clinic. The superior performance of PI3K PROTAC was also confirmed in lapatinib-resistant breast cancer xenograft tumors and patient-derived breast cancer organoids (PDOs). In conclusion, this study reveals that the novel PI3K PROTAC we synthesized could serve as an effective agent to overcome lapatinib resistance.
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Neoplasias da Mama , Proliferação de Células , Classe I de Fosfatidilinositol 3-Quinases , Resistencia a Medicamentos Antineoplásicos , Lapatinib , Proteólise , Receptor ErbB-2 , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Lapatinib/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Animais , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Camundongos , Proteólise/efeitos dos fármacos , Mutação , Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Camundongos Nus , TiazóisRESUMO
The NLRP3 inflammasome plays an important role in protecting the host from infection and aseptic inflammation, and its regulatory mechanism is not completely understood. Dysregulation of NLRP3 can cause diverse inflammatory diseases. HECTD3 is a E3 ubiquitin ligase of the HECT family that has been reported to participate in autoimmune and infectious diseases. However, the relationship between HECTD3 and the NLRP3 inflammasome has not been well studied. Herein, we show that HECTD3 blocks the interaction between NEK7 and NLRP3 to inhibit NLRP3 inflammasome assembly and activation. In BMDMs, Hectd3 deficiency promotes the assembly and activation of NLRP3 inflammasome and the secretion of IL-1ß, while the overexpression of HECTD3 inhibits these processes. Unexpectedly, HECTD3 functions in an E3 activity independent manner. Mechanically, the DOC domain of HECTD3 interacts with NACHT/LRR domain of NLRP3, which blocks NLRP3-NEK7 interaction and NLRP3 oligomerization. Furthermore, HECTD3 inhibits monosodium urate crystals (MSU)-induced gouty arthritis, a NLRP3-related disease. Thus, we reveal a novel regulatory mechanism of NLRP3 by HECTD3 and suggest HECTD3 could be a potential therapeutic target for NLRP3-dependent pathologies.
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Artrite Gotosa , Inflamassomos , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Inflamação , Interleucina-1beta , Quinases Relacionadas a NIMA/genéticaRESUMO
Background: Immune checkpoint blockers (ICBs) are revolutionized therapeutic strategies for cancer, but most patients with solid neoplasms remain resistant to ICBs, partly because of the difficulty in reversing the highly immunosuppressive tumor microenvironment (TME). Exploring the strategies for tumor immunotherapy is highly dependent on the discovery of molecular mechanisms of tumor immune escape and potential therapeutic target. Krüppel-like Factor 5 (KLF5) is a cell-intrinsic oncogene to promote tumorigenesis. However, the cell-extrinsic effects of KLF5 on suppressing the immune response to cancer remain unclear. Methods: We analyzed the immunosuppressive role of KLF5 in mice models transplanted with KLF5-deleted/overexpressing tumor cells. We performed RNA sequencing, immunohistochemistry, western blotting, real time-PCR, ELISA, luciferase assay, chromatin immunoprecipitation (ChIP), and flow cytometry to demonstrate the effects of KLF5 on CD8+ T cell infiltration and related molecular mechanism. Single-cell RNA sequencing and spatial transcriptomics analysis were applied to further decipher the association between KLF5 expression and infiltrating immune cells. The efficacy of KLF5/COX2 inhibitors combined with anti-programmed cell death protein 1 (anti-PD1) therapy were explored in pre-clinical models. Finally, a gene-expression signature depending on KLF5/COX2 axis and associated immune markers was created to predict patient survival. Results: KLF5 inactivation decelerated basal-like breast tumor growth in a CD8+ T-cell-dependent manner. Transcriptomic profiling revealed that KLF5 loss in tumors increases the number and activated function of T lymphocytes. Mechanistically, KLF5 binds to the promoter of the COX2 gene and promotes COX2 transcription; subsequently, KLF5 deficiency decreases prostaglandin E2 (PGE2) release from tumor cells by reducing COX2 expression. Inhibition of the KLF5/COX2 axis increases the number and functionality of intratumoral antitumor T cells to synergize the antitumorigenic effects of anti-PD1 therapy. Analysis of patient datasets at single-cell and spatial resolution shows that low expression of KLF5 is associated with an immune-supportive TME. Finally, we generate a KLF5/COX2-associated immune score (KC-IS) to predict patient survival. Conclusions: Our results identified a novel mechanism responsible for KLF5-mediated immunosuppression in TME, and targeting the KLF5/COX2/PGE2 axis is a critical immunotherapy sensitizer.
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Dinoprostona , Fatores de Transcrição , Camundongos , Animais , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Fatores de Transcrição/metabolismo , Imunoterapia , Linfócitos T CD8-Positivos , Microambiente Tumoral , Linhagem Celular Tumoral , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismoRESUMO
Triple-negative breast cancer (TNBC) has higher molecular heterogeneity and metastatic potential and the poorest prognosis. Because of limited therapeutics against TNBC, irradiation (IR) therapy is still a common treatment option for patients with lymph nodes or brain metastasis. Thus, it is urgent to develop strategies to enhance the sensitivity of TNBC tumors to low-dose IR. Here, the authors report that E3 ubiquitin ligase Ring finger protein 126 (RNF126) is important for IR-induced ATR-CHK1 pathway activation to enhance DNA damage repair (DDR). Mechanistically, RNF126 physically associates with the MRE11-RAD50-NBS1 (MRN) complex and ubiquitinates MRE11 at K339 and K480 to increase its DNA exonuclease activity, subsequent RPA binding, and ATR phosphorylation, promoting sustained DDR in a homologous recombination repair-prone manner. Accordingly, depletion of RNF126 leads to increased genomic instability and radiation sensitivity in both TNBC cells and mice. Furthermore, it is found that RNF126 expression is induced by IR activating the HER2-AKT-NF-κB pathway and targeting RNF126 expression with dihydroartemisinin significantly improves the sensitivity of TNBC tumors in the brain to IR treatment in vivo. Together, these results reveal that RNF126-mediated MRE11 ubiquitination is a critical regulator of the DDR, which provides a promising target for improving the sensitivity of TNBC to radiotherapy.
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Dano ao DNA , Reparo do DNA , Neoplasias de Mama Triplo Negativas , Ubiquitina-Proteína Ligases , Animais , Humanos , Camundongos , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Proteína Homóloga a MRE11/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/radioterapia , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Metastasis is the leading cause of cancer-related death. The interactions between circulating tumor cells and endothelial adhesion molecules in distant organs is a key step during extravasation in hematogenous metastasis. Surgery is a common intervention for most primary solid tumors. However, surgical trauma-related systemic inflammation facilitates distant tumor metastasis by increasing the spread and adhesion of tumor cells to vascular endothelial cells (ECs). Currently, there are no effective interventions to prevent distant metastasis. Here, we show that HECTD3 deficiency in ECs significantly reduces tumor metastasis in multiple mouse models. HECTD3 depletion downregulates expression of adhesion molecules, such as VCAM-1, ICAM-1 and E-selectin, in mouse primary ECs and HUVECs stimulated by inflammatory factors and inhibits adhesion of tumor cells to ECs both in vitro and in vivo. We demonstrate that HECTD3 promotes stabilization, nuclear localization and kinase activity of IKKα by ubiquitinating IKKα with K27- and K63-linked polyubiquitin chains at K296, increasing phosphorylation of histone H3 to promote NF-κB target gene transcription. Knockout of HECTD3 in endothelium significantly inhibits tumor cells lung colonization, while conditional knockin promotes that. IKKα kinase inhibitors prevented LPS-induced pulmonary metastasis. These findings reveal the promotional role of the HECTD3-IKKα axis in tumor hematogenous metastasis and provide a potential strategy for tumor metastasis prevention.
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Células Endoteliais , Neoplasias , Animais , Células Endoteliais/metabolismo , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Basal-like breast cancer (BLBC) accounts for approximately 15% of all breast cancer cases, and patients with BLBC have a low survival rate. Our previous study demonstrated that the KLF5 transcription factor promotes BLBC cell proliferation and tumor growth. In this study, we demonstrated that the histone deacetylase inhibitors (HDACi), suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA), increased KLF5 acetylation at lysine 369 (K369), downregulated KLF5 protein expression levels, and decreased cell viability in BLBC cell lines. HDACi target KLF5 for proteasomal degradation by promoting KLF5 protein ubiquitination. K369 acetylation of KLF5 decreases the binding between KLF5 and its deubiquitinase, BAP1. These findings revealed a novel mechanism by which HDACi suppress BLBC, and a novel crosstalk between KLF5 protein acetylation and ubiquitination.
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Neoplasias da Mama , Inibidores de Histona Desacetilases , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Ubiquitinação , Vorinostat/farmacologiaRESUMO
OBJECTIVES: It remains debatable if early mobilization (EM) yields a better clinical outcome than the late mobilization (LM) in adults with an acute and displaced distal radial fracture (DRF) of open reduction internal fixation (ORIF). Therefore, we aimed to perform a systematic review and meta-analysis of randomized controlled trials (RCTs), comparing clinical results with the safety of EM with LM following ORIF. METHODS: Databases such as Medline, Cochrane Central Register, and Embase were searched from Jan 1, 2000, to July 31, 2021, and RCTs comparing EM with LM for DRF with ORIF were included in the analysis. The primary outcome of study included disabilities of the Arm, Shoulder, and Hand (DASH) score at different follow-up times. Wherever the secondary outcomes included patient-rated wrist evaluation (PRWE), grip strength (GS), visual analog scale (VAS), wrist range of motion (WROM), and associated complications, the two independent reviewers did data extraction for the analysis. Effect sizes of outcome for each group were pooled using random-effects models; thereafter, the results were represented in the forest plots. RESULTS: Nine RCTs with 293 EM and 303 LM participants were identified and included in the study. Our analysis showed that the DASH score of the EM group was significantly better than LM group at the six weeks postoperatively (- 10.15; 95% CI - 15.74 to - 4.57, P < 0.01). Besides, the EM group also had better outcomes in PRWE, GS and WROM at 6 weeks. However, EM showed potential higher rate for implant loosening and/or fracture re-displacement complication (3.00; 95% CI 1.02-8.83, P = 0.05). CONCLUSION: Functionally, at earlier stages, EM for patients with DRF of ORIF may have a beneficial effect than LM. The mean differences in the DASH score at 6 weeks surpassed the minimal clinically important difference; however, the potentially higher risk of implant loosening and/or fracture re-displacement cannot be ignored. Due to the lack of definitive evidence, multicenter and large sample RCTs are required for determining the optimal rehabilitation protocol for DRF with ORIF. PROSPERO registration number: CRD42021240214 2021/2/28.
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Fraturas do Rádio , Placas Ósseas , Deambulação Precoce , Fixação Interna de Fraturas , Humanos , Estudos Multicêntricos como Assunto , Redução Aberta , Fraturas do Rádio/diagnóstico por imagem , Fraturas do Rádio/cirurgia , Amplitude de Movimento Articular , Resultado do TratamentoRESUMO
BACKGROUND: The objective of this systematic review was to evaluate current studies available reporting the antibiotic spacer combined with Ilizarov methods in the treatment of infected nonunion of tibia and to perform meta-analysis of bone results and infection recurrence to assess the efficacy of an antibiotic spacer combined with Ilizarov methods. METHODS: The MEDLINE, Embase, Cochrane Library, CNKI, and CBM (Chinese Biological Medicine) databases were searched for articles published between January 2000 and July 2020. Assessment of study quality was performed using a modified version of the Newcastle-Ottawa scale. Effect size and 95% confidence intervals were calculated for the main outcome. Heterogeneity was assessed. Fixed-effect modeling and Stata version 15.1 were used to analyze the data. Sensitivity analyses were conducted with the evidence of heterogeneity. RESULTS: 11 studies involving 210 patients with infected nonunion of tibia were finally included in our meta-analysis. Bone results and infection recurrence were analyzed based on the single-arm meta-analysis. The average of external fixation index (EFI) was 46.88 days/cm in all studies included. The excellent rate in bone results and the rate of infection recurrence was 65% (95% CI: [0.22, 0.97], I 2 = 0.0%, P = 0.932) and 6.99% (95% CI: [0.052, 0.325], I 2 = 0.0%, P = 1.000) in patients with infected nonunion of tibia treated with an antibiotic spacer combined with Ilizarov methods. CONCLUSIONS: Our meta-analysis revealed that the patients with infected nonunion of tibia treated with an antibiotic spacer combined with Ilizarov methods had a high rate of excellent bone results and a low rate of infection recurrence. Therefore, combining the antibiotic spacer with Ilizarov methods may be an applicable choice for repairing and reconstructing infected nonunion of tibia.
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Antibacterianos , Doenças Ósseas Infecciosas , Fraturas não Consolidadas/cirurgia , Técnica de Ilizarov , Fraturas da Tíbia/cirurgia , Adulto , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Doenças Ósseas Infecciosas/tratamento farmacológico , Doenças Ósseas Infecciosas/epidemiologia , Doenças Ósseas Infecciosas/cirurgia , Implantes de Medicamento , Feminino , Consolidação da Fratura , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Tíbia/cirurgia , Adulto JovemRESUMO
Increasing evidence suggested that a number of ubiquitin enzymes, including ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, E3 ubiquitin ligases and deubiquitination enzymes contribute to therapeutic resistance in triple-negative breast cancer (TNBC) cells. Inhibition of these enzymes with small molecule inhibitors may restore therapeutic sensitivity. Here, we demonstrated ubiquitin conjugating enzyme UbcH5b strongly supports HECTD3 auto-ubiquitination in vitro. Based on this, we developed a Fluorescence Resonance Energy Transfer (FRET) assay and identified three Schisandraceae triterpenoids, including PC3-15, to block HECTD3/UbcH5b auto-ubiquitination. Furthermore, we revealed that PC3-15 directly binds to UbcH5b and also inhibits UbcH5b-mediated p62 ubiquitination. We found that the UbcH5b-p62 axis confers TNBC cells resistance to lapatinib by promoting autophagy. Consistently, PC3-15 inhibits lapatinib-induced autophagy and increases lapatinib sensitivity in TNBC in vitro and in mouse xenografts. These findings suggest that the UbcH5b-p62 axis provides potential therapeutic targets and that Schisandraceae triterpenoids may be used for TNBC treatment in combination with lapatinib.
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Antineoplásicos/farmacologia , Lapatinib/farmacologia , Schisandra/química , Neoplasias de Mama Triplo Negativas/patologia , Triterpenos/farmacologia , Enzimas de Conjugação de Ubiquitina/efeitos dos fármacos , Animais , Humanos , Camundongos , Relação Estrutura-Atividade , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/enzimologia , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ubiquitin modification plays significant roles in protein fate determination, signaling transduction, and cellular processes. Over the past 2 decades, the number of studies on ubiquitination has demonstrated explosive growth. E3 ubiquitin ligases are the key enzymes that determine the substrate specificity and are involved in cancer. Several recent studies shed light on the functions and mechanisms of HECTD3 E3 ubiquitin ligase. This review describes the progress in the recent studies of HECTD3 in cancer and other diseases. We propose that HECTD3 is a potential biomarker and a therapeutic target, and discuss the future directions for HECTD3 investigations.
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Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ubiquitina-Proteína Ligases/análise , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitinação/efeitos dos fármacosRESUMO
The Krüppel-like factor 5 (KLF5) transcription factor is highly expressed in basal type breast cancer and promotes breast cancer cell proliferation, survival, migration, and tumorigenesis. KLF5 protein stability is regulated by ubiquitination. In this study, ubiquitin-specific protease 3 (USP3) was identified as a new KLF5 deubiquitinase by genome-wide siRNA library screening. We demonstrated that USP3 interacts with KLF5 and stabilizes KLF5 via deubiquitination. USP3 knockdown inhibits breast cancer cell proliferation in vitro and tumorigenesis in vivo, which can be partially rescued by ectopic expression of KLF5. Furthermore, we observed a positive correlation between USP3 and KLF5 protein expression levels in human breast cancer samples. These findings suggest that USP3 is a new KLF5 deubiquitinase and that USP3 may represent a potential therapeutic target for breast cancer.
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Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Células HEK293 , Humanos , Camundongos Nus , Ligação Proteica , Estabilidade Proteica , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Lysine-63-linked (K63-linked) polyubiquitination of TRAF3 coordinates the engagement of pattern-recognition receptors with recruited adaptor proteins and downstream activator TBK1 in pathways that induce type I IFN. Whether autoubiquitination or other E3 ligases mediate K63-linked TRAF3 polyubiquitination remains unclear. We demonstrated that mice deficient in the E3 ligase gene Hectd3 remarkably increased host defense against infection by intracellular bacteria Francisella novicida, Mycobacterium, and Listeria by limiting bacterial dissemination. In the absence of HECTD3, type I IFN response was impaired during bacterial infection both in vivo and in vitro. HECTD3 regulated type I IFN production by mediating K63-linked polyubiquitination of TRAF3 at residue K138. The catalytic domain of HECTD3 regulated TRAF3 K63 polyubiquitination, which enabled TRAF3-TBK1 complex formation. Our study offers insights into mechanisms of TRAF3 modulation and provides potential therapeutic targets against infections by intracellular bacteria and inflammatory diseases.
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Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Interferon Tipo I/biossíntese , Fator 3 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Modelos Animais de Doenças , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/metabolismo , Feminino , Francisella , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Listeriose/imunologia , Listeriose/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 3 Associado a Receptor de TNF/química , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
It is well documented that hypoxia activates the hypoxia-inducible factor 1-alpha (HIF1α)/vascular endothelial growth factor A (VEGFA) axis to promote angiogenesis in breast cancer. However, it is unclear how this axis is negatively regulated. In this study, we demonstrated that miR-153 directly inhibits expression of HIF1α by binding to the 3'UTR of HIF1A mRNA, as well as suppresses tube formation of primary human umbilical vein endothelial cells (HUVECs) and breast cancer angiogenesis by decreasing the secretion of VEGFA. Importantly, expression of miR-153 was induced by hypoxia-stimulated ER stress, which activates IRE1α and its downstream transcription factor X-box binding protein 1 (XBP1). X-box binding protein 1 directly binds to the promoter of the miR-153 host gene PTPRN and activates transcription. These results indicate that hypoxia induces miR-153 to fine tune the HIF1α/VEGFA axis in breast cancer angiogenesis and miR-153 could be used for breast cancer anti-angiogenesis therapy.
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Neoplasias da Mama , Endorribonucleases/fisiologia , MicroRNAs/genética , Neovascularização Patológica/genética , Proteínas Serina-Treonina Quinases/fisiologia , Hipóxia Tumoral/genética , Proteína 1 de Ligação a X-Box/fisiologia , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células Cultivadas , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Transdução de Sinais/genética , Regulação para Cima/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Out of the breast cancer subtypes, triple-negative breast cancer (TNBC) has the poorest prognosis without effective targeted therapies. Metformin, a first-line drug for type 2 diabetes mellitus, was demonstrated to target breast cancer stem cells selectively. However, the efficiency and the mechanism of action of metformin in TNBC are unclear. In this study, we demonstrated that metformin decreased the percentage of TNBC stem cells partially through the downregulation of the expression of the stem cell transcription factor Krüppel-like factor 5 (KLF5) and its downstream target genes, such as Nanog and FGF-BP1, in TNBC cell lines. Metformin induced glycogen synthase kinase-3ß (GSK3ß)-mediated KLF5 protein phosphorylation and degradation through the inhibition of protein kinase A (PKA) activity in TNBC cells. Consistently, PKA activators increased the expression levels of KLF5. We observed a positive correlation between p-CREB, p-GSK3ß, KLF5 and FGF-BP1 protein levels in human TNBC samples. These findings suggest that metformin suppresses TNBC stem cells partially through the PKA-GSK3ß-KLF5 signaling pathway.
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Pulse wave imaging (PWI) is an ultrasound-based method to visualize the propagation of pulse wave and to quantitatively estimate regional pulse wave velocity (PWV) of the arteries within the imaging field of view (FOV). To guarantee the reliability of PWV measurement, high frame rate imaging is required, which can be achieved by reducing the line density of ultrasound imaging or transmitting plane wave at the expense of spatial resolution and/or signal-to-noise ratio (SNR). In this study, a composite, full-view imaging method using motion matching was proposed with both high temporal and spatial resolution. Ultrasound radiofrequency (RF) data of 4 sub-sectors, each with 34 beams, including a common beam, were acquired successively to achieve a frame rate of â¼507 Hz at an imaging depth of 35 mm. The acceleration profiles of the vessel wall estimated from the common beam were used to reconstruct the full-view (38-mm width, 128-beam) image sequence. The feasibility of mapping local PWV variation along the artery using PWI technique was preliminarily validated on both homogeneous and inhomogeneous polyvinyl alcohol (PVA) cryogel vessel phantoms. Regional PWVs for the three homogeneous phantoms measured by the proposed method were in accordance with the sparse imaging method (38-mm width, 32-beam) and plane wave imaging method. Local PWV was estimated using the above-mentioned three methods on 3 inhomogeneous phantoms, and good agreement was obtained in both the softer (1.91±0.24 m/s, 1.97±0.27 m/s and 1.78±0.28 m/s) and the stiffer region (4.17±0.46 m/s, 3.99±0.53 m/s and 4.27±0.49 m/s) of the phantoms. In addition to the improved spatial resolution, higher precision of local PWV estimation in low SNR circumstances was also obtained by the proposed method as compared with the sparse imaging method. The proposed method might be helpful in disease detections through mapping the local PWV of the vascular wall.
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Análise de Onda de Pulso/métodos , Estudos de Viabilidade , Humanos , Imagens de Fantasmas , Razão Sinal-Ruído , Ultrassonografia/métodosRESUMO
The tree shrew is becoming an attractive experimental animal model for human breast cancer owing to a closer relationship to primates/humans than rodents. Tree shrews are superior to classical primates because tree shrew are easier to manipulate, maintain and propagate. It is required to establish a high-efficiency tree shrew breast cancer model for etiological research and drug assessment. Our previous studies suggest that 7,12-dimethylbenz(a)anthracene (DMBA) and medroxyprogesterone acetate (MPA) induce breast tumors in tree shrews with a low frequency (<50%) and long latency (â¼ 7-month), making these methods less than ideal. We induced mammary tumors in tree shrew (Tupaia belangeri chinensis) by injection of lentivirus expressing the PyMT oncogene into mammary ducts of 22 animals. Most tree shrews developed mammary tumors with a latency of about three weeks, and by 7 weeks all injected tree shrews had developed mammary tumors. Among these, papillary carcinoma is the predominant tumor type. One case showed lymph node and lung metastasis. Interestingly, the expression levels of phosphorylated AKT, ERK and STAT3 were elevated in 41-68% of PyMT-induced mammary tumors, but not all tumors. Finally, we observed that the growth of PyMT-induced tree shrew mammary tumors was significantly inhibited by Cisplatin and Epidoxorubicin. PyMT-induced tree shrew mammary tumor model may be suitable for further breast cancer research and drug development, due to its high efficiency and short latency.
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Antígenos Virais de Tumores/genética , Modelos Animais de Doenças , Neoplasias Mamárias Animais/etiologia , Polyomavirus/imunologia , Tupaiidae , Animais , Carcinoma Papilar/etiologia , Células Epiteliais/patologia , Receptor alfa de Estrogênio/análise , Feminino , Lentivirus/genética , Neoplasias Mamárias Animais/química , Neoplasias Mamárias Animais/tratamento farmacológico , Fator de Transcrição STAT3/metabolismoRESUMO
Growing evidence suggests that YAP/TAZ are mediators of the Hippo pathway and promote breast cancer. However, the roles of YAP/TAZ transcription factor partners TEADs in breast cancer remain unclear. Here we found that TEAD4 was expressed in breast cancer cell lines, especially in triple negative breast cancers (TNBC) cell lines. TEAD4 binds to KLF5. Knockdown of either TEAD4 or KLF5 in HCC1937 and HCC1806 cells induced the expression of CDK inhibitor p27. Depletion of either TEAD4 or KLF5 activated the p27 gene promoter and increased the p27 mRNA levels. Depletion of p27 partially prevents growth inhibition caused by TEAD4 and KLF5 knockdown. TEAD4 overexpression stimulated proliferation in vitro and tumor growth in mice, while stable knockdown of TEAD4 inhibited proliferation in vitro and tumor growth in mice. Thus TEAD4 and KLF5, in collaboration, promoted TNBC cell proliferation and tumor growth in part by inhibiting p27 gene transcription. TEAD4 is a potential target and biomarker for the development of novel therapeutics for breast cancer.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Musculares/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Feminino , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase , Fatores de Transcrição de Domínio TEA , Transcrição Gênica , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
BACKGROUND: Open injuries of the Achilles tendon, which can be complicated by skin and bone injuries, continue to be a great challenge for surgeons. This study aims to report our experience with treatment of open Achilles tendon defects, focusing on the injury mechanisms, soft tissue coverage and late complications. METHODS: A retrospective review was performed on 31 patients with open Achilles tendon defects between 1999 and 2011. The analyzed factors were injury mechanisms, surgeries, and long follow-up complications. The defect lengths of the Achilles tendons in the study ranged from 1 to 11 cm and the soft tissue defects ranged from 3 × 3 to 12 × 10 cm. Nine types of flaps were used for the coverage of concomitant skin defects. RESULTS: Motorcycle spoke injuries were the most common cause of injury. There was no complete flap loss or rerupture of the reconstructed Achilles tendon. At the latest follow-up, all limbs were preserved and all the patients had regained full walking abilities. The algorithm of one-stage reconstruction was established, according to the defect length of the Achilles tendon and the defect size of skin. Late complications included maximum dorsiflexion loss and failure of heel raising ability on the single reconstructed foot. CONCLUSION: Open Achilles tendon defects are characteristic of concurrent skin and bone injuries and the reconstruction protocols of the different tissues should not be separated.