RESUMO
Hippo signaling controls organ size and tumor progression through a conserved pathway leading to nuclear translocation of the transcriptional effector Yki/Yap/Taz. Most of our understanding of Hippo signaling pertains to its cytoplasmic regulation, but how the pathway is controlled in the nucleus remains poorly understood. Here we uncover an evolutionarily conserved mechanism by which CDK7 promotes Yki/Yap/Taz stabilization in the nucleus to sustain Hippo pathway outputs. We found that a modular E3 ubiquitin ligase complex CRL4DCAF12 binds and targets Yki/Yap/Taz for ubiquitination and degradation, whereas CDK7 phosphorylates Yki/Yap/Taz at S169/S128/S90 to inhibit CRL4DCAF12 recruitment, leading to Yki/Yap/Taz stabilization. As a consequence, inactivation of CDK7 reduced organ size and inhibited tumor growth, which could be reversed by restoring Yki/Yap activity. Our study identifies an unanticipated layer of Hippo pathway regulation, defines a novel mechanism by which CDK7 regulates tissue growth, and implies CDK7 as a drug target for Yap/Taz-driven cancer.
Assuntos
Carcinogênese/genética , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinogênese/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes/genética , Drosophila melanogaster/genética , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/fisiopatologia , Camundongos , Tamanho do Órgão/genética , Fenilenodiaminas/farmacologia , Proteólise , Pirimidinas/farmacologia , Proteínas de Sinalização YAP , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Inhibition of CDK7 is a promising strategy for cancer therapy. CDK7 so far has been understood mainly in the context of Pol II-driven transcription. However, how are the roles of CDK7 in the "basal" transcriptional machinery reconciled with the function of CDK7 as inducer of specific transcriptional programs in tumor cells? In this issue of Genes & Development, Cho and colleagues (pp. 53-71) advance in this direction, demonstrating that attenuation of CDK7 fosters the oncogenic activity of the YAP/TAZ/Yki coactivators. CDK7 directly phosphorylates YAP/TAZ/Yki in the nucleus, protecting them from ubiquitination and degradation, in a manner independent from the Hippo cascade and independent from CDK7 basal transcriptional functions.
Assuntos
Neoplasias , Fatores de Transcrição , Proteínas Adaptadoras de Transdução de Sinal , Via de Sinalização Hippo , Humanos , Tamanho do Órgão , Fosfoproteínas , Proteínas Serina-Treonina QuinasesRESUMO
The role of transfer RNA (tRNA)-derived fragment (tRF) in various diseases has been established. However, the effect of tRF-3023b on inflammation remains unclear. Inflammation was imitated in RAW264.7 cells by adding Lipopolysaccharide (LPS). Cells were first divided into control, LPS, and LPS + Bulleyaconitine A (BLA) groups. The contents of TNF-α, IL-6, and MCP-1 were quantified using ELISA. The levels of cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), and the phosphorylation of nuclear factor-kappa B (NF-κB)-P65 (p-P65) were detected by Western blotting. RNA sequencing was utilized to find differentially expressed tRFs (DE-tRFs) among three groups. The levels of various tRFs were checked by quantitative real-time PCR (qRT-PCR). Cell cycle and apoptosis were checked by flow cytometry. Dluciferase reporter assay was applied to predict and confirm the interaction between tRF-3023b and Cullin 4A (Cul4a), subsequently RNA pull-down followed by mass spectrometry analysis were conducted. BLA treatment decreased the contents of TNF-α, IL-6, MCP-1, and the expression levels of COX2, iNOS, p-P65. We found 6 DE-tRFs in LPS + BLA group compared to LPS group, tRF-3023b was high expression in control and BLA groups, and the lowest in LPS group. Cul4a was a direct target of tRF-3023b. tRF-3023b mimic affected the cell cycle distribution, promoted cells apoptosis, and suppressed the TNF-α, IL-6, MCP-1, COX2, iNOS and p-P65. The suppression of Cul4a affected the cell cycle distribution, resulted in an increase of cell apoptosis while a decrease of TNF-α, IL-6, MCP-1, COX2, iNOS and p-P65. Furthermore, Cul4a overexpression reversed the effect of tRF-3023b mimic. Cul4a knockdown reversed the effect of tRF-3023b inhibitor. Our study positions tRF-3023b as a compelling candidate, through its interaction with Cul4a, the underlying mechanism on inflammation maybe related to NF-κB pathway. The study provides a basis for exploring new therapeutic strategies for inflammation.
Assuntos
Proteínas Culina , NF-kappa B , Fator de Necrose Tumoral alfa , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-6/genética , Lipopolissacarídeos/toxicidade , NF-kappa B/genética , RNA de Transferência , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Animais , Camundongos , Células RAW 264.7 , Proteínas Culina/genética , Proteínas Culina/metabolismoRESUMO
IMPORTANCE: The human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are etiologic agents of numerous B cell lymphomas. A hallmark of gammaherpesvirus infection is their ability to establish lifelong latency in B cells. However, the specific mechanisms that mediate chronic infection in B cells in vivo remain elusive. Cellular E3 ubiquitin ligases regulate numerous biological processes by catalyzing ubiquitylation and modifying protein location, function, or half-life. Many viruses hijack host ubiquitin ligases to evade antiviral host defense and promote viral fitness. Here, we used the murine gammaherpesvirus 68 in vivo system to demonstrate that the E3 ligase Cul4b is essential for this virus to establish latency in germinal center B cells. These findings highlight an essential role for this E3 ligase in promoting chronic gammaherpesvirus infection in vivo and suggest that targeted inhibition of E3 ligases may provide a novel and effective intervention strategy against gammaherpesvirus-associated diseases.
Assuntos
Linfócitos B , Gammaherpesvirinae , Infecções por Herpesviridae , Infecção Persistente , Animais , Camundongos , Linfócitos B/enzimologia , Linfócitos B/metabolismo , Linfócitos B/virologia , Proteínas Culina/metabolismo , Gammaherpesvirinae/fisiologia , Centro Germinativo/citologia , Centro Germinativo/virologia , Infecções por Herpesviridae/enzimologia , Infecções por Herpesviridae/virologia , Infecção Persistente/enzimologia , Infecção Persistente/virologia , Ubiquitinas/metabolismo , Latência ViralRESUMO
Gastric cancer is a serious malignant tumor in the world, accounting for the third cause of cancer death worldwide. The pathogenesis of gastric cancer is very complex, in which epigenetic inheritance plays an important role. In our study, we found that DZIP3 was significantly up-regulated in gastric cancer tissues as compared to adjacent normal tissue, which suggested it may be play a crucial part in gastric cancer. To clarify the mechanism of it, we further analyzed the interacting proteome and transcriptome of DZIP3. An association between DZIP3 and some epigenetic regulators, such as CUL4B complex, was verified. We also present the first proteomic characterization of the protein-protein interaction (PPI) network of DZIP3. Then, the transcriptome analysis of DZIP3 demonstrated that knockdown DZIP3 increased a cohort of genes, including SETD7 and ZBTB4, which have essential role in tumors. We also revealed that DZIP3 promotes proliferation and metastasis of gastric cancer cells. And the higher expression of DZIP3 is positively associated with the poor prognosis of several cancers. In summary, our study revealed a mechanistic role of DZIP3 in promoting proliferation and metastasis in gastric cancer, supporting the pursuit of DZIP3 as a potential target for gastric cancer therapy.
Assuntos
Neoplasias Gástricas , Humanos , Neoplasias Gástricas/patologia , Proteômica , Proliferação de Células/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Movimento Celular/genética , Metástase Neoplásica , Histona-Lisina N-Metiltransferase/genética , Proteínas de Ligação a RNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Culina/metabolismoRESUMO
Schlafen-11 (SLFN11) inactivation in â¼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1-CUL4CDT2 ubiquitin ligase.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Dano ao DNA/genética , Replicação do DNA , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteólise , Mutações Sintéticas Letais/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/metabolismo , Cromossomos Humanos/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Estabilidade Enzimática , Genoma Humano , Humanos , Mitose , Modelos Biológicos , Terapia de Alvo Molecular , Fosforilação , Ligação Proteica , Interferência de RNA , Transdução de SinaisRESUMO
Circular RNA (circRNA) plays important roles in lumbar degenerative diseases. This study aimed to investigate the role of circSNTB2 in regulating the development of lumbar disc herniation (LDH) in vitro and in vivo. The abnormally expressed circSNTB2 in intervertebral disc degeneration (IDD) through bioinformatics analysis was identified, and verified in nucleus pulposus (NP) tissues of patients with LDH. NP cells were treated with TNF-α to mimic the LDH microenvironment. RT-qPCR was applied to determine levels of mRNA and microRNA (miRNA) in clinical samples and cells. We performed CCK-8, EdU, TUNEL and flow cytometric apoptosis assays to evaluate the proliferation and apoptosis of NP cells. The predicted the miRNAs and downstream target genes were verified with the help of luciferase reporter gene and RNA pull-down experiments. Finally, we established an LDH rat model to further verify the role of circSNTB2 in vivo. circSNTB2 was significantly up-regulated in the NP tissues of LDH group and TNF-α -treated NP cells. miR-665 binds to circSNTB2 and cullin 4A (CUL4A) is the downstream target gene of miR-665. Knockdown of circSNTB2 promoted NP cells proliferation and inhibited apoptosis, which was reversed by down-regulation of miR-665. In addition, up-regulated CUL4A reversed the effects of over-expressed miR-665 on proliferation and apoptosis of NP cells. Meanwhile, results of in vivo experiments demonstrated that knocking down circSNTB2 alleviated LDH-induced thermo-mechanical pain and NP injury. In summary, circSNTB2 regulates the proliferation and apoptosis of NP by mediating miR-665 regulation of CUL4A, which provides a reliable idea for targeted therapy of LDH.
RESUMO
Cullin 4B (CUL4B), which acts as a scaffold protein in CUL4B-RING ubiquitin ligase complexes (CRL4B), is frequently overexpressed in cancer and represses tumor suppressors through epigenetic mechanisms. However, the expression and function of CUL4B in esophageal squamous cell carcinoma (ESCC) have not been well illustrated. In this study, we show that upregulation of CUL4B in ESCC cells enhances proliferation, invasion and cisplatin (CDDP)-resistance, while knockdown of CUL4B significantly represses the malignant activities. Mechanistically, we demonstrate that CUL4B promotes proliferation and migration of ESCC cells through inhibiting expression of transforming growth factor beta receptor III (TGFBR3). CRL4B complex binds to the promoter of TGFBR3, and represses its transcription by catalyzing monoubiquitination at H2AK119 and coordinating with PRC2 and HDAC complexes. Taken together, our findings establish a critical role for the CUL4B/TGFBR3 axis in the regulation of ESCC malignancy.
Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Humanos , Carcinoma de Células Escamosas do Esôfago/genética , Neoplasias Esofágicas/genética , Proteínas Culina/genética , Proteínas Culina/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fenótipo , Proliferação de Células/fisiologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Movimento Celular/genéticaRESUMO
In maize (Zea mays L.), lateral roots are formed in the differentiation zone of all root types in a multi-step process. The maize mutant lateral rootless 1 (lrt1) is defective in lateral root formation in primary and seminal roots but not in shoot-borne roots. We cloned the lrt1 gene by mapping in combination with BSA-seq and subsequent validation via CRISPR/Cas9. The lrt1 gene encodes a 209 kDa homolog of the DDB1-CUL4-ASSOCIATED FACTOR (DCAF) subunit of the CUL4-based E3 ubiquitin ligase (CRL4) complex localized in the nucleus. DDB1-CUL4-ASSOCIATED FACTOR proteins are encoded by an evolutionary old gene family already present in nonseed plants. They are adaptors that bind substrate proteins and promote their ubiquitylation, thus typically marking them for subsequent degradation in the 26S proteasome. Gene expression studies demonstrated that lrt1 transcripts are expressed preferentially in the meristematic zone of all root types of maize. Downregulation of the rum1 gene in lrt1 mutants suggests that lrt1 acts upstream of the lateral root regulator rum1. Our results demonstrate that DCAF proteins play a key role in root-type-specific lateral root formation in maize. Together with its role in nitrogen acquisition in nitrogen-poor soil, lrt1 could be a promising target for maize improvement.
Assuntos
Ubiquitina-Proteína Ligases , Zea mays , Zea mays/genética , Zea mays/metabolismo , Subunidades Proteicas/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Meristema/metabolismoRESUMO
CUL4A is an ubiquitin ligase deregulated in numerous pathologies including cancer and even hijacked by viruses for facilitating their survival and propagation. However, its role in Human papilloma virus (HPV)-mediated cervical carcinogenesis remains elusive. The UALCAN and GEPIA datasets were analyzed to ascertain the transcript levels of CUL4A in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) patients. Subsequently, various biochemical assays were employed to explore the functional contribution of CUL4A in cervical carcinogenesis and to shed some light on its involvement in Cisplatin resistance in cervical cancer. Our UALCAN and GEPIA datasets analyses reveal elevated CUL4A transcript levels in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) patients that correlate with adverse clinicopathological parameters such as tumor stage and lymph node metastasis. Kaplan-Meier plot and GEPIA assessment depict poor prognosis of CESC patients having high CUL4A expression. Varied biochemical assays illustrate that CUL4A inhibition severely curtails hallmark malignant properties such as cellular proliferation, migration, and invasion of cervical cancer cells. We also show that CUL4A knockdown in HeLa cells causes increased susceptibility and better apoptotic induction toward Cisplatin, a mainstay drug used in cervical cancer treatment. More interestingly, we find reversion of Cisplatin-resistant phenotype of HeLa cells and an augmented cytotoxicity towards the platinum compound upon CUL4A downregulation. Taken together, our study underscores CUL4A as a cervical cancer oncogene and illustrates its potential as a prognosis indicator. Our investigation provides a novel avenue in improving current anti-cervical cancer therapy and overcoming the bottle-neck of Cisplatin resistance.
RESUMO
Circular RNA (circRNA) regulates malignant tumors, including ovarian cancer (OC). The present research study aimed to reveal the biological mechanism of circRNA mitofusin 2 (circMFN2) in OC. Cell biological behaviors were investigated using clonogenicity assay, EdU assay, transwell assay, and flow cytometry analysis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analysis were implemented to detect the levels of circMFN2, miR-198, Cullin 4B (CUL4B), and apoptosis-related proteins. Glycolysis was assessed by glucose assay kit, lactate assay kit, and ATP level detection kit. The relationships among miR-198, circMFN2, and CUL4B were verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. The xenograft mice model was used to analyze tumor growth in vivo. The expression of circMFN2 and CUL4B was increased, while miR-330-5p was decreased in OC tissues or cells. The absence of CircMFN2 hindered cell proliferation, migration, invasion, and glycolysis and promoted apoptosis in OC cells. We found that circMFN2 promoted CUL4B expression via sponging miR-198. MiR-198 depletion reversed circMFN2 knockdown-induced effects in OC cells. Furthermore, CUL4B overexpression overturned the inhibitory effect of miR-198 in OC cells. And the absence of circMFN2 inhibited tumor growth in vivo. CircMFN2 repressed OC progression by regulating the miR-198/CUL4B axis.
Assuntos
MicroRNAs , Neoplasias Ovarianas , Humanos , Animais , Camundongos , Feminino , RNA Circular/genética , Neoplasias Ovarianas/genética , Glicólise , Proliferação de Células , Modelos Animais de Doenças , Ácido Láctico , MicroRNAs/genética , Linhagem Celular Tumoral , Proteínas Culina/genéticaRESUMO
Understanding the molecular mechanisms underlying osteoclast differentiation provides insights into bone loss and even osteoporosis. The specific mechanistic actions of cullin 4A (CUL4A) in osteoclast differentiation and resultant osteoporosis is poorly explored. We developed a mouse model of osteoporosis using bilateral ovariectomy (OVX) and examined CUL4A expression. It was noted that CUL4A expression was increased in the bone marrow of OVX mice. Overexpression of CUL4A promoted osteoclast differentiation, and knockdown of CUL4A alleviated osteoporosis symptoms of OVX mice. Bioinformatic analyses were applied to identify the downstream target genes of microRNA-340-5p (miR-340-5p), followed by interaction analysis. The bone marrow macrophages (BMMs) were isolated from femur of OVX mice, which were transfected with different plasmids to alter the expression of CUL4A, Zinc finer E-box binding homeobox 1 (ZEB1), miR-340-5p, and Toll-like receptor 4 (TLR4). ChIP assay was performed to detect enrichment of ZEB1 promoter by H3K4me3 antibody in BMMs. ZEB1 was overexpressed in the bone marrow of OVX mice. Overexpression of CUL4A mediated H3K4me3 methylation to increase ZEB1 expression, thus promoting osteoclast differentiation. Meanwhile, ZEB1 could inhibit miR-340-5p expression and upregulate HMGB1 to induce osteoclast differentiation. Overexpressed ZEB1 activated the TLR4 pathway by regulating the miR-340-5p/HMGB1 axis to induce osteoclast differentiation, thus promoting the development of osteoporosis. Overall, E3 ubiquitin ligase CUL4A can upregulate ZEB1 to repress miR-340-5p expression, leading to HMGB1 upregulation and the TLR4 pathway activation, which promotes osteoclast differentiation and the development of osteoporosis.
Assuntos
Proteína HMGB1 , MicroRNAs , Osteoporose , Feminino , Camundongos , Animais , Receptor 4 Toll-Like/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Regulação para Cima , Osteoporose/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Diferenciação Celular , Osteogênese/genéticaRESUMO
Deciphering how DCAFs (DDB1-CUL4 Associated Factors) modulate a broad spectrum of cellular processes, including cell cycle progression and maintenance of genomic integrity is critical to better understand cellular homeostasis and diseases. Cells contain more than 100 DCAFs that associate with the Cullin-Ring Ubiquitin Ligase 4 (CRL4) complex that target specific protein substrates for degradation. DCAFs are thought to act as substrate receptors that dictate the specificity of the ubiquitination machinery ("catalytic DCAFs"). However, recent studies have suggested that some DCAFs might play a different role by targeting CRL4 complexes to distinct cellular compartments ("structural DCAFs"). Once localized to their correct cellular domains, these CRLs dissociate from the structural DCAFs prior to their association with other, substrate-specific catalytic DCAFs. Thus, we propose that DCAF switches can provide a mechanistic basis for the degradation of proteins that regulate cell growth and proliferation at precise points in space and time.
Assuntos
Proteínas de Ligação a DNA , Ubiquitina-Proteína Ligases , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Circular RNA lysine demethylase 4A (circKDM4A) is also named circ_0012098 and its abnormal expression has been confirmed in serum exosomes of prostate cancer (PC) patients. However, whether PC progression involves the exosomal circ_0012098 remains unknown. RNA expression of circKDM4A, microRNA-338-3p (miR-338-3p) and cullin 4B (CUL4B) was detected by quantitative real-time polymerase chain reaction. Protein expression was checked by Western blot. The positive expression rate of nuclear proliferation marker (ki-67) was analyzed by immunohistochemistry assay. Dual-luciferase reporter assay and RNA immunoprecipitation assay were used to identify the interaction between miR-338-3p and circKDM4A or CUL4B. Mouse model assay was performed to determine the effect of exosomal circKDM4A on tumorigenesis in vivo. CircKDM4A expression was significantly upregulated in the serum exosomes from PC patients compared with the exosomes from healthy volunteers. Exosomes treatment promoted the proliferation, migration and invasion of PC cells but inhibited apoptosis; however, these effects were attenuated after circKDM4A knockdown. Meanwhile, circKDM4A depletion restored exosome-increased circKDM4A expression. Additionally, circKDM4A acted as a miR-338-3p sponge, and miR-338-3p bound to CUL4B in PC cells. CircKDM4A regulated the effect of exosome-induced PC cell malignancy by interacting with miR-338-3p and CUL4B. Moreover, circKDM4A silencing relieved exosome-induced tumor growth in vivo. Exosomal circKDM4A promoted PC malignant progression by the miR-338-3p/CUL4B axis, providing a therapeutic target for PC.
Assuntos
MicroRNAs , Neoplasias da Próstata , Animais , Camundongos , Masculino , Humanos , Neoplasias da Próstata/genética , Carcinogênese , Apoptose , Modelos Animais de Doenças , MicroRNAs/genética , Proliferação de Células , Linhagem Celular Tumoral , Proteínas Culina/genéticaRESUMO
Platinum-based chemotherapy drugs play a very important role in the treatment of patients with advanced colorectal cancer, but the drug resistance of platinum-based chemotherapy drugs is an important topic that puzzles us. If we can find mechanisms of resistance, it will be revolutionary for us. We analysed the differential genes, core genes and their enrichment pathways in platinum-resistant and non-resistant patients through a public database. Platinum-resistant cell lines were cultured in vitro for in vitro colony and Transwell analysis. Tumorigenesis analysis of nude mice in vivo. Verify the function of core genes. Through differential gene and enrichment analysis, we found that CUL4B was the main factor affecting platinum drug resistance and EMT. Our hypothesis was further verified by in vitro drug-resistant and wild-type cell lines and in vivo tumorigenesis analysis of nude mice. CUL4B leads to platinum drug resistance in colorectal cancer by affecting tumour EMT.
Assuntos
Neoplasias Colorretais , Resistencia a Medicamentos Antineoplásicos , Compostos de Platina , Animais , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese , Transformação Celular Neoplásica , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Resistência a Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Camundongos Nus , Compostos de Platina/farmacologia , Compostos de Platina/uso terapêuticoRESUMO
The aryl hydrocarbon receptor (AHR) is a transcription factor activated by exogenous halogenated polycyclic aromatic hydrocarbon compounds, including the environmental toxin TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and naturally occurring dietary and endogenous compounds. The activated AHR enhances transcription of specific genes including phase I and phase II metabolism enzymes and other targets genes such as the TCDD-inducible poly(ADP-ribose) polymerase (TiPARP). The regulation of AHR activation is a dynamic process: immediately after transcriptional activation of the AHR by TCDD, the AHR is exported from the nucleus to the cytoplasm where it is subjected to proteasomal degradation. However, the mechanisms regulating AHR degradation are not well understood. Here, we studied the role of two enzymes reported to enhance AHR breakdown: the cullin 4B (CUL4B)AHR complex, an E3 ubiquitin ligase that targets the AHR and other proteins for ubiquitination, and TiPARP, which targets proteins for ADP-ribosylation, a posttranslational modification that can increase susceptibility to degradation. Using a WT mouse embryonic fibroblast (MEF) cell line and an MEF cell line in which CUL4B has been deleted (MEFCul4b-null), we discovered that loss of CUL4B partially prevented AHR degradation after TCDD exposure, while knocking down TiPARP in MEFCul4b-null cells completely abolished AHR degradation upon TCDD treatment. Increased TCDD-activated AHR protein levels in MEFCul4b-null and MEFCul4b-null cells in which TiPARP was knocked down led to enhanced AHR transcriptional activity, indicating that CUL4B and TiPARP restrain AHR action. This study reveals a novel function of TiPARP in controlling TCDD-activated AHR nuclear export and subsequent proteasomal degradation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Culina/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Células Cultivadas , Poluentes Ambientais/toxicidade , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Camundongos , ProteóliseRESUMO
Acute respiratory distress syndrome (ARDS) is a critical disease with a high mortality rate, characterized by obstinate hypoxemia caused by accumulation of alveolar fluid and excessive uncontrolled inflammation. Na,K-ATPase α1 (ATP1A1) subunit is an important component of Na,K-ATPase that transports Na+ and K+ and scavenges alveolar fluid. The function of Na,K-ATPase is always impaired during ARDS and results in more severe symptoms of ARDS. However, the regulatory mechanism of Na,K-ATPase after ARDS remains unclear. Here, we revealed ATP1A1 was downregulated post-transcriptionally by an E3 ligase component CUL4B mediated proteasomal degradation. Moreover, we found insulin could inhibit the upregulation of CUL4B in an insulin receptor cofactor HCF-1-dependent manner. Our study resolved the molecular mechanism underlying the clearance impairment of alveolar fluid and provided a clue for the usage of insulin as a potential therapeutic medicine for ARDS.
Assuntos
Proteínas Culina , Síndrome do Desconforto Respiratório , ATPase Trocadora de Sódio-Potássio , Proteínas Culina/metabolismo , Humanos , Insulina/metabolismo , Lipopolissacarídeos/metabolismo , Alvéolos Pulmonares/metabolismo , Síndrome do Desconforto Respiratório/tratamento farmacológico , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in plants. A key regulatory system for protein stability is given by the CULLIN-RING E3 ligases (CRLs). In this work, MYB43 is identified as a novel target of a CUL4-DDB1-PRL1 (PLEIOTROPIC REGULATORY LOCUS 1)-RING E3 ligase (CRL4PRL1 E3 ligase). Its stability depends on the presence of PRL1, a WD40-containing protein functioning as a substrate receptor of the CRL4 E3 ligases. Genetic studies have indicated that MYB43 is a negative regulator of cadmium (Cd) tolerance in Arabidopsis by transcriptional inhibition of important Cd transporters (HMA2, HMA3 and HMA4), while PRL1 and CUL4 positively regulate Cd tolerance. Expression of CUL4 and PRL1 was enhanced in response to Cd stress, and PRL1 can interact with and target MYB43 for degradation depending on assembly of CRL4PRL1 E3 ligase, and consequently increase the expression of HMA2, HMA3 and HMA4 through attenuating the transcriptional inhibition. HMA2 and HMA4 are shown to transport cadmium ion (Cd2+ ) from the roots of plants to the shoots through the xylem, ultimately increasing the plants' tolerance to Cd stress.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Adaptação Biológica/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cádmio/metabolismo , Cádmio/toxicidade , Proteínas de Transporte/metabolismo , Genes de Plantas/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Glioma multiforme (GBM) is the most common malignant primary brain tumors. Despite the considerable advances in GBM treatment, it is still one of the most lethal forms of brain tumor. New clinical biomarkers and therapeutic targets are immediately required. MicroRNAs (miRNAs) are a class of small, evolutionarily conserved noncoding RNAs and have emerged as the key regulators of many cancers. Here in this study, we showed that miR-674-5p was probably an important regulator of glioma cell growth. After the transfection with miR-674-5p mimic or inhibitor, we found that the expression level of miR-674-5p was negatively related with cell proliferation and migration in C6 cells. Based on the prediction of the target genes of miR-674-5p on the website, we chose Cullin 4B (Cul4b), a gene upregulated in GBM, and proved that it was a target of miR-674-5p. In addition, we explored the role of miR-674-5p in glioma growth in vivo. Taken together, the present study indicated that miR-674-5p suppressed glioma cell proliferation and migration by targeting Cul4b.
Assuntos
Neoplasias Encefálicas , Proteínas Culina , Glioma , MicroRNAs , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proteínas Culina/genética , Proteínas Culina/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RatosRESUMO
Tamoxifen (TAM) resistance is a significant clinical challenge in endocrine therapies for estrogen receptor (ER)-positive breast cancer patients. Cullin 4B (CUL4B), which acts as a scaffold protein in CUL4B-RING ubiquitin ligase complexes (CRL4B), is frequently overexpressed in cancer and represses tumor suppressors through diverse epigenetic mechanisms. However, the role and the underlying mechanisms of CUL4B in regulating drug resistance remain unknown. Here, we showed that CUL4B promotes TAM resistance in breast cancer cells through a miR-32-5p/ER-α36 axis. We found that upregulation of CUL4B correlated with decreased TAM sensitivity of breast cancer cells, and knockdown of CUL4B or expression of a dominant-negative CUL4B mutant restored the response to TAM in TAM-resistant MCF7-TAMR and T47D-TAMR cells. Mechanistically, we demonstrated that CUL4B renders breast cancer cells TAM-resistant by upregulating ER-α36 expression, which was mediated by downregulation of miR-32-5p. We further showed that CRL4B epigenetically represses the transcription of miR-32-5p by catalyzing monoubiquitination at H2AK119 and coordinating with PRC2 and HDAC complexes to promote trimethylation at H3K27 at the promoter of miR-32-5p. Pharmacologic or genetic inhibition of CRL4B/PRC2/HDAC complexes significantly increased TAM sensitivity in breast cancer cells in vitro and in vivo. Taken together, our findings thus establish a critical role for the CUL4B-miR-32-5p-ER-α36 axis in the regulation of TAM resistance and have important therapeutic implications for combined application of TAM and the inhibitors of CRL4B/PRC2/HDAC complex in breast cancer treatment. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.