RESUMO
The Warburg effect is a hallmark of cancer that refers to the preference of cancer cells to metabolize glucose anaerobically rather than aerobically1,2. This results in substantial accumulation of lacate, the end product of anaerobic glycolysis, in cancer cells3. However, how cancer metabolism affects chemotherapy response and DNA repair in general remains incompletely understood. Here we report that lactate-driven lactylation of NBS1 promotes homologous recombination (HR)-mediated DNA repair. Lactylation of NBS1 at lysine 388 (K388) is essential for MRE11-RAD50-NBS1 (MRN) complex formation and the accumulation of HR repair proteins at the sites of DNA double-strand breaks. Furthermore, we identify TIP60 as the NBS1 lysine lactyltransferase and the 'writer' of NBS1 K388 lactylation, and HDAC3 as the NBS1 de-lactylase. High levels of NBS1 K388 lactylation predict poor patient outcome of neoadjuvant chemotherapy, and lactate reduction using either genetic depletion of lactate dehydrogenase A (LDHA) or stiripentol, a lactate dehydrogenase A inhibitor used clinically for anti-epileptic treatment, inhibited NBS1 K388 lactylation, decreased DNA repair efficacy and overcame resistance to chemotherapy. In summary, our work identifies NBS1 lactylation as a critical mechanism for genome stability that contributes to chemotherapy resistance and identifies inhibition of lactate production as a promising therapeutic cancer strategy.
Assuntos
Proteínas de Ciclo Celular , Resistencia a Medicamentos Antineoplásicos , Ácido Láctico , Proteínas Nucleares , Reparo de DNA por Recombinação , Animais , Feminino , Humanos , Masculino , Camundongos , Hidrolases Anidrido Ácido/metabolismo , Anaerobiose , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Instabilidade Genômica , Ácido Láctico/metabolismo , Lisina/química , Lisina/metabolismo , Lisina Acetiltransferase 5/metabolismo , Lisina Acetiltransferase 5/genética , Proteína Homóloga a MRE11/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Organoides , Glicólise , Terapia Neoadjuvante , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/deficiência , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Anticonvulsivantes/farmacologiaRESUMO
Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61 remains unclear. Here, we found that CYR61 is important for proper cell cycle progression. Specifically, CYR61 interacts with microtubules and promotes microtubule polymerization to ensure mitotic entry. Moreover, CYR61 interacts with PLK1 and accumulates during the mitotic process, followed by degradation as mitosis concludes. The proteolysis of CYR61 requires the PLK1 kinase activity, which directly phosphorylates two conserved motifs on CYR61, enhancing its interaction with the SCF E3 complex subunit FBW7 and mediating its degradation by the proteasome. Mutations of phosphorylation sites of Ser167 and Ser188 greatly increase CYR61's stability, while deletion of CYR61 extends prophase and metaphase and delays anaphase onset. In summary, our findings highlight the precise control of the intracellular CYR61 by the PLK1-FBW7 pathway, accentuating its significance as a microtubule-associated protein during mitotic progression.
Assuntos
Proteínas de Ciclo Celular , Proteína Rica em Cisteína 61 , Microtúbulos , Mitose , Quinase 1 Polo-Like , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Humanos , Mitose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteína Rica em Cisteína 61/metabolismo , Proteína Rica em Cisteína 61/genética , Microtúbulos/metabolismo , Proteína 7 com Repetições F-Box-WD/metabolismo , Proteína 7 com Repetições F-Box-WD/genética , Células HeLa , Fosforilação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
Breast cancers involving mutations in homologous recombination (HR) genes, most commonly BRCA1 and BRCA2 (BRCA1/2), respond well to PARP inhibitors and platinum-based chemotherapy. However, except for these specific HR genes, it is not clear which other mutations contribute to homologous recombination defects (HRD). Here, we performed next-generation sequencing of tumor tissues and matched blood samples from 119 breast cancer patients using the OncoScreen Plus panel. Genomic mutation characteristics and HRD scores were analyzed. In the HR genes, we found that BRCA1/2 and PLAB2 mutations were related to HRD. HRD was also detected in a subset of patients without germline or somatic mutations in BRCA1/2, PLAB2, or other HR-related genes. Notably, LRP1B, NOTCH3, GATA2, and CARD11 (abbreviated as LNGC) mutations were associated with high HRD scores in breast cancer patients. Furthermore, functional experiments demonstrated that silencing CARD11 and GATA2 impairs HR repair efficiency and enhances the sensitivity of tumor cells to olaparib treatment. In summary, in the absence of mutations in the HR genes, the sensitivity of tumor cells to PARP inhibitors and platinum-based chemotherapy may be enhanced in a subset of breast cancer patients with LNGC somatic mutations.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Mutação , Recombinação HomólogaRESUMO
The cell cycle is a highly regulated process in which proteins involved in cell cycle progression exhibit periodic expression patterns, controlled by specific mechanisms such as transcription, translation, and degradation. However, the precise mechanisms underlying the oscillations of mRNA levels in cell cycle regulators are not fully understood. In this study, we observed that the stability of cyclin D1 (CCND1) mRNA fluctuates during the cell cycle, with increased stability during interphase and decreased stability during the M phase. Additionally, we identified a key RNA binding protein, positive coactivator 4 (PC4), which plays a crucial role in stabilizing CCND1 mRNA and regulating its periodic expression. Moreover, the binding affinity of PC4 to CCND1 mRNA is modulated by two cell cycle-specific posttranslational modifications: ubiquitination of K68 enhances binding and stabilizes the CCND1 transcript during interphase, while phosphorylation of S17 inhibits binding during the M phase, leading to degradation of CCND1 mRNA. Remarkably, PC4 promotes the transition from G1 to S phase in the cell cycle, and depletion of PC4 enhances the efficacy of CDK4/6 inhibitors in hepatocellular carcinoma, suggesting that PC4 could serve as a potential therapeutic target. These findings provide valuable insights into the intricate regulation of cell cycle dynamics.
Assuntos
Ciclo Celular , Ciclina D1 , Estabilidade de RNA , Proteínas de Ligação a RNA , Ciclo Celular/genética , Divisão Celular , Ciclina D1/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina , Estabilidade de RNA/genética , RNA Mensageiro/genética , Masculino , Animais , Camundongos , Camundongos Endogâmicos BALB C , Humanos , Linhagem Celular Tumoral , Proteínas de Ligação a RNA/genética , Fosforilação , UbiquitinaçãoRESUMO
Poly(ADP-ribosyl)ation (PARylation) is a critical posttranslational modification that plays a vital role in maintaining genomic stability via a variety of molecular mechanisms, including activation of replication stress and the DNA damage response. The nudix hydrolase NUDT16 was recently identified as a phosphodiesterase that is responsible for removing ADP-ribose units and that plays an important role in DNA repair. However, the roles of NUDT16 in coordinating replication stress and cell cycle progression remain elusive. Here, we report that SETD3, which is a member of the SET-domain containing protein (SETD) family, is a novel substrate for NUDT16, that its protein levels fluctuate during cell cycle progression, and that its stability is strictly regulated by NUDT16-mediated dePARylation. Moreover, our data indicated that the E3 ligase CHFR is responsible for the recognition and degradation of endogenous SETD3 in a PARP1-mediated PARylation-dependent manner. Mechanistically, we revealed that SETD3 associates with BRCA2 and promotes its recruitment to stalled replication fork and DNA damage sites upon replication stress or DNA double-strand breaks, respectively. Importantly, depletion of SETD3 in NUDT16-deficient cells did not further exacerbate DNA breaks or enhance the sensitivity of cancer cells to IR exposure, suggesting that the NUDT16-SETD3 pathway may play critical roles in the induction of tolerance to radiotherapy. Collectively, these data showed that NUDT16 functions as a key upstream regulator of SETD3 protein stability by reversing the ADP-ribosylation of SETD3, and NUDT16 participates in the resolution of replication stress and facilitates HR repair.
Assuntos
ADP-Ribosilação , Neoplasias , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Neoplasias/genética , Neoplasias/radioterapia , Poli(ADP-Ribose) Polimerase-1/genética , Processamento de Proteína Pós-Traducional , Humanos , Linhagem Celular , Pirofosfatases/genética , Pirofosfatases/metabolismo , Histona Metiltransferases/genética , Histona Metiltransferases/metabolismoRESUMO
Activation of innate immunity in the brain is a prominent feature of Alzheimer's disease (AD). The present study investigated the regulation of innate immunity by wild-type serum injection in a transgenic AD mouse model. We found that treatment with wild-type mouse serum significantly reduced the number of neutrophils and microglial reactivity in the brains of APP/PS1 mice. Mimicking this effect, neutrophil depletion via Ly6G neutralizing antibodies resulted in improvements in AD brain functions. Serum proteomic analysis identified vascular endothelial growth factor-A (VEGF-A) and chemokine (C-X-C motif) ligand 1 (CXCL1) as factors enriched in serum samples, which are crucial for neutrophil migration and chemotaxis, leukocyte migration, and cell chemotaxis. Exogenous VEGF-A reversed amyloid ß (Aß)-induced decreases in cyclin-dependent kinase 5 (Cdk5) and increases in CXCL1 in vitro and blocked neutrophil infiltration into the AD brain. Endothelial Cdk5 overexpression conferred an inhibitory effect on CXCL1 and neutrophil infiltration, thereby restoring memory abilities in APP/PS1 mice. Our findings uncover a previously unknown link between blood-derived VEGF signaling and neutrophil infiltration and support targeting endothelial Cdk5 signaling as a potential therapeutic strategy for AD.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Camundongos , Animais , Camundongos Transgênicos , Fator A de Crescimento do Endotélio Vascular , Infiltração de Neutrófilos , Proteômica , Doença de Alzheimer/terapia , Transtornos da Memória , Modelos Animais de Doenças , Precursor de Proteína beta-Amiloide/genética , Presenilina-1/genéticaRESUMO
Aberrant SUMOylation contributes to the progression of hepatocellular carcinoma (HCC), yet the molecular mechanisms have not been well elucidated. RING-type E3 ubiquitin ligase RNF146 is a key regulator of the Wnt/ß-catenin signaling pathway, which is frequently hyperactivated in HCC. Here, it is identified that RNF146 can be modified by SUMO3. By mutating all lysines in RNF146, we found that K19, K61, K174 and K175 are the major sites for SUMOylation. UBC9/PIAS3/MMS21 and SENP1/2/6 mediated the conjugation and deconjugation of SUMO3, respectively. Furthermore, SUMOylation of RNF146 promoted its nuclear localization, while deSUMOylation induced its cytoplasmic localization. Importantly, SUMOylation promotes the association of RNF146 with Axin to accelerate the ubiquitination and degradation of Axin. Intriguingly, only UBC9/PIAS3 and SENP1 can act at K19/K175 in RNF146 and affect its role in regulating the stability of Axin. In addition, inhibiting RNF146 SUMOylation suppressed the progression of HCC both in vitro and in vivo. And, patients with higher expression of RNF146 and UBC9 have the worst prognosis. Taken together, we conclude that RNF146 SUMOylation at K19/K175 promotes its association with Axin and accelerates Axin degradation, thereby enhancing ß-catenin signaling and contributing to cancer progression. Our findings reveal that RNF146 SUMOylation is a potential therapeutic target in HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Proteína Axina/genética , Proteína Axina/metabolismo , beta Catenina/metabolismo , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Chaperonas Moleculares/metabolismo , Proteínas Inibidoras de STAT Ativados , Sumoilação , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização WntRESUMO
Exosomes are well-known key mediators of intercellular communication and contribute to various physiological and pathological processes. Their biogenesis involves four key steps, including cargo sorting, MVB formation and maturation, transport of MVBs, and MVB fusion with the plasma membrane. Each process is modulated through the competition or coordination of multiple mechanisms, whereby diverse repertoires of molecular cargos are sorted into distinct subpopulations of exosomes, resulting in the high heterogeneity of exosomes. Intriguingly, cancer cells exploit various strategies, such as aberrant gene expression, posttranslational modifications, and altered signaling pathways, to regulate the biogenesis, composition, and eventually functions of exosomes to promote cancer progression. Therefore, exosome biogenesis-targeted therapy is being actively explored. In this review, we systematically summarize recent progress in understanding the machinery of exosome biogenesis and how it is regulated in the context of cancer. In particular, we highlight pharmacological targeting of exosome biogenesis as a promising cancer therapeutic strategy.
Assuntos
Exossomos , Neoplasias , Humanos , Exossomos/metabolismo , Corpos Multivesiculares/metabolismo , Neoplasias/metabolismo , Comunicação Celular , Membrana Celular/metabolismoRESUMO
Chromosome rearrangement plays important roles in development, carcinogenesis and evolution. However, its mechanism and subsequent effects are not fully understood. Large-scale chromosome rearrangement has been performed in the simple eukaryote, wine yeast, but the relative research in mammalian cells remains at the level of individual chromosome rearrangement due to technical limitations. In this study, we used CRISPR-Cas9 to target the highly repetitive human endogenous retrotransposons, LINE-1 and Alu, resulting in a large number of DNA double-strand breaks in the chromosomes. While this operation killed the majority of the cells, we eventually obtained live cell groups. Karyotype analysis and genome re-sequencing proved that we have achieved global chromosome rearrangement (GCR) in human cells. The copy number variations of the GCR genomes showed typical patterns observed in tumor genomes. The ATAC-seq and RNA-seq further revealed that the epigenetic and transcriptomic landscapes were deeply reshaped by GCR. Gene expressions related to p53 pathway, DNA repair, cell cycle and apoptosis were greatly altered to facilitate the cell survival. Our study provided a new application of CRISPR-Cas9 and a practical approach for GCR in complex mammalian genomes.
Assuntos
Edição de Genes , Transcriptoma , Sistemas CRISPR-Cas , Cromossomos/metabolismo , Variações do Número de Cópias de DNA , Edição de Genes/métodos , Genoma Humano , Humanos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
MEX3A is an RNA-binding protein that mediates mRNA decay through binding to 3' untranslated regions. However, its role and mechanism in clear cell renal cell carcinoma remain unknown. In this study, we found that MEX3A expression was transcriptionally activated by ETS1 and upregulated in clear cell renal cell carcinoma. Silencing MEX3A markedly reduced clear cell renal cell carcinoma cell proliferation in vitro and in vivo. Inhibiting MEX3A induced G1/S cell-cycle arrest. Gene set enrichment analysis revealed that E2F targets are the central downstream pathways of MEX3A. To identify MEX3A targets, systematic screening using enhanced cross-linking and immunoprecipitation sequencing, and RNA-immunoprecipitation sequencing assays were performed. A network of 4,000 genes was identified as potential targets of MEX3A. Gene ontology analysis of upregulated genes bound by MEX3A indicated that negative regulation of the cell proliferation pathway was highly enriched. Further assays indicated that MEX3A bound to the CDKN2B 3' untranslated region, promoting its mRNA degradation. This leads to decreased levels of CDKN2B and an uncontrolled cell cycle in clear cell renal cell carcinoma, which was confirmed by rescue experiments. Our findings revealed that MEX3A acts as a post-transcriptional regulator of abnormal cell-cycle progression in clear cell renal cell carcinoma.
RESUMO
Ring-finger protein 126 (RNF126), an E3 ubiquitin ligase, plays crucial roles in various biological processes, including cell proliferation, DNA damage repair, and intracellular vesicle trafficking. Whether RNF126 is modulated by posttranslational modifications is poorly understood. Here, we show that PARP1 interacts with and poly(ADP)ribosylates RNF126, which then recruits the PAR-binding E3 ubiquitin ligase CHFR to promote ubiquitination and degradation of RNF126. Moreover, RNF126 is required for the activation of ATR-Chk1 signaling induced by either irradiation (IR) or a PARP inhibitor (PARPi), and depletion of RNF126 increases the sensitivity of triple-negative breast cancer (TNBC) cells to PARPi treatment. Our findings suggest that PARPi-mediated upregulation of RNF126 protein stability contributes to TNBC cell resistance to PARPi. Therefore, targeting the E3 ubiquitin ligase RNF126 may be a novel treatment for overcoming the resistance of TNBC cells to PARPi in clinical trials.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Neoplasias/metabolismo , Ftalazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ubiquitina-Proteína Ligases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Radiation resistance is a major cause of lung cancer treatment failure. Armadillo (ARM) superfamily proteins participate in various fundamental cellular processes; however, whether ARM proteins regulate radiation resistance is not fully understood. Here, we used an unbiased CRISPR/Cas9 library screen and identified plakophilin 2 (PKP2), a member of the ARM superfamily of proteins, as a critical driver of radiation resistance in lung cancer. The PKP2 level was significantly higher after radiotherapy than before radiotherapy, and high PKP2 expression after radiotherapy predicted poor overall survival (OS) and postprogression survival (PPS). Mechanistically, mass spectrometry analysis identified that PKP2 was methylated at the arginine site and interacted with protein arginine methyltransferase 1 (PRMT1). Methylation of PKP2 by PRMT1 stabilized ß-catenin by recruiting USP7, further inducing LIG4, a key DNA ligase in nonhomologous end-joining (NHEJ) repair. Concomitantly, PKP2-induced radioresistance depended on facilitating LIG4-mediated NHEJ repair in lung cancer. More strikingly, after exposure to irradiation, treatment with the PRMT1 inhibitor C-7280948 abolished PKP2-induced radioresistance, and C-7280948 is a potential radiosensitizer in lung cancer. In summary, our results demonstrate that targeting the PRMT1/PKP2/ß-catenin/LIG4 pathway is an effective approach to overcome radiation resistance in lung cancer.
Assuntos
Sistemas CRISPR-Cas/genética , Neoplasias Pulmonares/genética , Placofilinas/metabolismo , Radiossensibilizantes/uso terapêutico , beta Catenina/metabolismo , Humanos , Neoplasias Pulmonares/patologiaRESUMO
The pro-inflammatory and pro-fibrotic liver microenvironment facilitates hepatocarcinogenesis. However, the effects and mechanisms by which the hepatic fibroinflammatory microenvironment modulates intrahepatic hepatocellular carcinoma (HCC) progression and its response to systematic therapy remain largely unexplored. We established a syngeneic orthotopic HCC mouse model with a series of persistent liver injury induced by CCl4 gavage, which mimic the dynamic effect of hepatic pathology microenvironment on intrahepatic HCC growth and metastasis. Non-invasive bioluminescence imaging was applied to follow tumour progression over time. The effect of the liver microenvironment modulated by hepatic injury on sorafenib resistance was investigated in vivo and in vitro. We found that the persistent liver injury facilitated HCC growth and metastasis, which was positively correlated with the degree of liver inflammation rather than the extent of liver fibrosis. The inflammatory cytokines in liver tissue were clearly increased after liver injury. The two indicated cytokines, tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6), both promoted intrahepatic HCC progression via STAT3 activation. In addition, the hepatic inflammatory microenvironment contributed to sorafenib resistance through the anti-apoptotic protein mediated by STAT3, and STAT3 inhibitor S3I-201 significantly improved sorafenib efficacy impaired by liver inflammation. Clinically, the increased inflammation of liver tissues was accompanied with the up-regulated STAT3 activation in HCC. Above all, we concluded that the hepatic inflammatory microenvironment promotes intrahepatic HCC growth, metastasis and sorafenib resistance through activation of STAT3.
Assuntos
Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Microambiente Celular , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Humanos , Mediadores da Inflamação , Cirrose Hepática/complicações , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Sorafenibe/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hypoxia causes a series of responses supporting cells to survive in harsh environments. Substantial post-transcriptional and translational regulation during hypoxia has been observed. However, detailed regulatory mechanism in response to hypoxia is still far from complete. RNA m6A modification has been proven to govern the life cycle of RNAs. Here, we reported that total m6A level of mRNAs was decreased during hypoxia, which might be mediated by the induction of m6A eraser, ALKBH5. Meanwhile, expression levels of most YTH family members of m6A readers were systematically down-regulated. Transcriptome-wide analysis of m6A revealed a drastic reprogramming of m6A epitranscriptome during cellular hypoxia. Integration of m6A epitranscriptome with either RNA-seq based transcriptome analysis or mass spectrometry (LC-MS/MS) based proteome analysis of cells upon hypoxic stress revealed that reprogramming of m6A epitranscriptome reshaped the transcriptome and proteome, thereby supporting efficient generation of energy for adaption to hypoxia. Moreover, ATP production was blocked when silencing an m6A eraser, ALKBH5, under hypoxic condition, demonstrating that m6A pathway is an important regulator during hypoxic response. Collectively, our studies indicate that crosstalk between m6A and HIF1 pathway is essential for cellular response to hypoxia, providing insights into the underlying molecular mechanisms during hypoxia.
Assuntos
Adenosina/análogos & derivados , Epigênese Genética , Hipóxia/genética , Hipóxia/metabolismo , Proteoma , Transcriptoma , Adenosina/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida , Biologia Computacional/métodos , Epigenômica/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Ontologia Genética , Humanos , Proteômica/métodos , Estresse Fisiológico/genética , Espectrometria de Massas em TandemRESUMO
Ewing sarcoma (ES) is a type of highly aggressive pediatric tumor in bones and soft tissues and its metastatic spread remains the most powerful predictor of poor outcome. We previously identified that the transcription factor hepatoma-derived growth factor (HDGF) promotes ES tumorigenesis. However, the mechanisms underlying ES metastasis remain unclear. Here, we show that HDGF drives ES metastasis in vitro and in vivo, and HDGF reduces metastasis-free survival (MFS) in two independent large cohorts of human ES patients. Integrative analyses of HDGF ChIP-seq and gene expression profiling in ES cells reveal that HDGF regulates multiple metastasis-associated genes, among which activated leukocyte cell adhesion molecule (ALCAM) emerges as a major HDGF target and a novel metastasis-suppressor in ES. HDGF down-regulates ALCAM, induces expression and activation of the downstream effectors Rho-GTPase Rac1 and Cdc42, and promotes actin cytoskeleton remodeling and cell-matrix adhesion. In addition, repression of ALCAM and activation of Rac1 and Cdc42 are required for the pro-metastatic functions of HDGF in vitro. Moreover, analyses in murine models with ES tumor orthotopic implantation and experimental metastasis, as well as in human ES samples, demonstrate the associations among HDGF, ALCAM, and GTPases expression levels. Furthermore, high HDGF/low ALCAM expression define a subgroup of patients harboring the worst MFS. These findings suggest that the HDGF/ALCAM/GTPases axis represents a promising therapeutic target for limiting ES metastasis.
Assuntos
Antígenos CD/fisiologia , Neoplasias Ósseas/patologia , Moléculas de Adesão Celular Neuronais/fisiologia , Proteínas Fetais/fisiologia , GTP Fosfo-Hidrolases/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Sarcoma de Ewing/patologia , Citoesqueleto de Actina/química , Adolescente , Adulto , Animais , Linhagem Celular Tumoral , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Transdução de Sinais/fisiologia , Adulto JovemRESUMO
Repair of DNA double-strand breaks (DSBs) is essential for genome integrity, and is accompanied by transcriptional repression at the DSB regions. However, the mechanisms how DNA repair induces transcriptional inhibition remain elusive. Here, it is identified that BRD7 participates in DNA damage response (DDR) and is recruited to the damaged chromatin via ATM signaling. Mechanistically, BRD7 joins the polycomb repressive complex 2 (PRC2), the nucleosome remodeling and histone deacetylation (NuRD) complex at the damaged DNA and recruits E3 ubiquitin ligase RNF168 to the DSBs. Furthermore, ATM-mediated BRD7 phosphorylation is required for recruitment of the PRC2 complex, NuRD complex, DSB sensor complex MRE11-RAD50-NBS1 (MRN), and RNF168 to the active transcription sites at DSBs, resulting in transcriptional repression and DNA repair. Moreover, BRD7 deficiency sensitizes cancer cells to PARP inhibition. Collectively, BRD7 is crucial for DNA repair and DDR-mediated transcription repression, which may serve as a therapeutic target. The findings identify the missing link between DNA repair and transcription regulation that maintains genome integrity.
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Rationale: Neoadjuvant chemotherapy has become the standard treatment of locally advanced breast cancer. Antimicrotubule drugs and DNA-damaging drugs are the most popular medicines used for neoadjuvant chemotherapy. However, we are unable to predict which chemotherapeutic drug will benefit to an individual patient. PARK2 as a tumor suppressor in breast cancer has been reported. While the role of PARK2 in chemotherapy response remains unknown. In this study, we explore the impact of PARK2 on chemosensitivity in breast cancer. Methods: PARK2 expression in breast cancer patients with different neoadjuvant chemotherapeutic regimens was studied using immunohistochemistry. Data was correlated to disease-free survival (DFS), overall survival and pathologic complete response (pCR). The functional roles of PARK2 were demonstrated by a series of in vitro and in vivo experiments. Including mass spectrometry, Co-immunoprecipitation, isolation of subcellular fractionation, fluorescence microscopy, in vivo ubiquitination assay and luciferase analyses. Results: Highly expressed PARK2 predicted better response to antimicrotubule drugs-containing regimen associated with higher rate of pathologic complete response (pCR). In contrast, PARK2 expression did not predict response to the DNA-damaging drugs regimen. Following antimicrotubule drugs treatment, levels of PARK2 was upregulated due to the repression of STAT3-mediated transcriptional inhibition of PARK2. Moreover, overexpression of PARK2 specifically rendered cells more sensitive to antimicrotubule drugs, but not to DNA-damaging drugs. Depletion of PARK2 enhanced resistance to antimicrotubule drugs. Mechanistically, PARK2 markedly activated the mitochondrial pathway of apoptosis after exposure to antimicrotubule drugs. This occurred through downregulating the antiapoptotic protein, phospho-BCL-2. BCL-2 phosphorylation can be specifically induced by antimicrotubule drugs, whereas DNA-damaging drugs do not. Notably, PARK2 interacted with phospho-BCL-2 (Ser70) and promoted ubiquitination of BCL-2 in an E3 ligase-dependent manner. Hence, PARK2 significantly enhanced the chemosensitivity of antimicrotubule drugs both in vitro and in vivo, while loss-of-function PARK2 mutants did not. Conclusions: Our findings explained why PARK2 selectively confers chemosensitivity to antimicrotubule drugs, but not to DNA-damaging drugs. In addition, we identified PARK2 as a novel mediator of antimicrotubule drugs sensitivity, which can predict response of breast cancer patients to antimicrotubule drugs-containing regime.
Assuntos
Antineoplásicos/farmacologia , Apoptose/fisiologia , Microtúbulos/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Intervalo Livre de Doença , Regulação para Baixo/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Células MCF-7 , Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/fisiologiaRESUMO
Rationale: The forkhead box A1 (FOXA1) is a crucial transcription factor in initiation and development of breast, lung and prostate cancer. Previous studies about the FOXA1 transcriptional network were mainly focused on protein-coding genes. Its regulatory network of long non-coding RNAs (lncRNAs) and their role in FOXA1 oncogenic activity remains unknown. Methods: The Cancer Genome Atlas (TCGA) data, RNA-seq and ChIP-seq data were used to analyze FOXA1 regulated lncRNAs. RT-qPCR was used to detect the expression of DSCAM-AS1, RT-qPCR and Western blotting were used to determine the expression of FOXA1, estrogen receptor α (ERα) and Y box binding protein 1 (YBX1). RNA pull-down and RIP-qPCR were employed to investigate the interaction between DSCAM-AS1 and YBX1. The effect of DSCAM-AS1 on malignant phenotypes was examined through in vitro and in vivo assays. Results: In this study, we conducted a global analysis of FOXA1 regulated lncRNAs. For detailed analysis, we chose lncRNA DSCAM-AS1, which is specifically expressed in lung adenocarcinoma, breast and prostate cancer. The expression level of DSCAM-AS1 is regulated by two super-enhancers (SEs) driven by FOXA1. High expression levels of DSCAM-AS1 was associated with poor prognosis. Knockout experiments showed DSCAM-AS1 was essential for the growth of xenograft tumors. Moreover, we demonstrated DSCAM-AS1 can regulate the expression of the master transcriptional factor FOXA1. In breast cancer, DSCAM-AS1 was also found to regulate ERα. Mechanistically, DSCAM-AS1 interacts with YBX1 and influences the recruitment of YBX1 in the promoter regions of FOXA1 and ERα. Conclusion: Our study demonstrated that lncRNA DSCAM-AS1 was transcriptionally activated by super-enhancers driven by FOXA1 and exhibited lineage-specific expression pattern. DSCAM-AS1 can promote cancer progression by interacting with YBX1 and regulating expression of FOXA1 and ERα.
Assuntos
Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/metabolismo , RNA Longo não Codificante/genética , Proteína 1 de Ligação a Y-Box/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/mortalidade , Adenocarcinoma de Pulmão/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sequenciamento de Cromatina por Imunoprecipitação , Biologia Computacional , Conjuntos de Dados como Assunto , Progressão da Doença , Elementos Facilitadores Genéticos/genética , Receptor alfa de Estrogênio/genética , Retroalimentação Fisiológica , Feminino , Técnicas de Inativação de Genes , Células HEK293 , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Prognóstico , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , RNA Longo não Codificante/metabolismo , RNA-Seq , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
NFYA (nuclear transcription factor Y, subunit A) is a CCAAT-binding transcription factor. Accumulating evidence suggests that NFYA plays an important role in breast, ovarian, lung and gastric cancer. However, the role of NFYA in clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, it was discovered that the expression of NFYA is elevated in tissues of ccRCC patient and high NFYA expression is linked to poor overall survival in ccRCC patient. Inhibition of G1/S cell cycle transition and decreased cell proliferation were observed upon NFYA knockdown in ccRCC cells. Moreover, further investigation revealed that NFYA binds directly to the promoter region of both CDK4 and cyclin D1 (CCND1) thus transactivating their expression, resulting in RB phosphorylation and the activation of subsequent E2F pathway activation. Taken together, these findings imply the oncogenic role of NFYA in ccRCC progression and its potential as a target for ccRCC therapy.