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1.
Cell Rep ; 38(5): 110304, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35108543

RESUMO

Cancer cells adapt to hypoxia through HIFs (hypoxia-inducible factors), which initiate the transcription of numerous genes for cancer cell survival in the hypoxia microenvironment. In this study, we find that the FACT (facilitates chromatin transcription) complex works cooperatively with HIFs to facilitate the expeditious expression of HIF targets for hypoxia adaptation. Knockout (KO) of the FACT complex abolishes HIF-mediated transcription by impeding transcription elongation in hypoxic cancer cells. Interestingly, the FACT complex is post-translationally regulated by PHD/VHL-mediated hydroxylation and proteasomal degradation, in similar fashion to HIF-1/2α. Metabolic tracing confirms that FACT KO suppresses glycolytic flux and impairs lactate extrusion, leading to intracellular acidification and apoptosis in cancer cells. Therapeutically, hepatic artery ligation and anti-angiogenic inhibitors adversely induce intratumoral hypoxia, while co-treatment with FACT inhibitor curaxin remarkably hinders the growth of hypoxic tumors. In summary, our findings suggest that the FACT complex is a critical component of hypoxia adaptation and a therapeutic target for hypoxic tumors.


Assuntos
Chaperonas de Histonas/metabolismo , Hipóxia/metabolismo , Oxigênio/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Hipóxia/genética
2.
Commun Biol ; 4(1): 888, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285329

RESUMO

Epigenetic deregulation plays an essential role in hepatocellular carcinoma (HCC) progression. Bromodomains are epigenetic "readers" of histone acetylation. Recently, bromodomain inhibitors have exhibited promising therapeutic potential for cancer treatment. Using transcriptome sequencing, we identified BRPF1 (bromodomain and PHD finger containing 1) as the most significantly upregulated gene among the 43 bromodomain-containing genes in human HCC. BRPF1 upregulation was significantly associated with poor patient survival. Gene ablation or pharmacological inactivation of BRPF1 significantly attenuated HCC cell growth in vitro and in vivo. BRPF1 was involved in cell cycle progression, senescence and cancer stemness. Transcriptome sequencing revealed that BRPF1 is a master regulator controlling the expression of multiple key oncogenes, including E2F2 and EZH2. We demonstrated that BRPF1 activated E2F2 and EZH2 expression by facilitating promoter H3K14 acetylation through MOZ/MORF complex. In conclusion, BRPF1 is frequently upregulated in human HCCs. Targeting BRPF1 may be an approach for HCC treatment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Hepatocelular/tratamento farmacológico , Proteínas de Ligação a DNA/genética , Neoplasias Hepáticas/tratamento farmacológico , Ativação Transcricional , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Carcinoma Hepatocelular/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C
3.
Gut ; 69(2): 329-342, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31439637

RESUMO

OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.


Assuntos
Carcinoma Hepatocelular/fisiopatologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Grupo de Alta Mobilidade/fisiologia , Chaperonas de Histonas/fisiologia , Neoplasias Hepáticas/fisiopatologia , Estresse Oxidativo/fisiologia , Fatores de Elongação da Transcrição/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carbazóis/farmacologia , Carbazóis/uso terapêutico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/prevenção & controle , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Inativação de Genes/métodos , Proteínas de Grupo de Alta Mobilidade/antagonistas & inibidores , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/fisiopatologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Camundongos Endogâmicos BALB C , Camundongos Nus , Estresse Oxidativo/genética , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Fatores de Elongação da Transcrição/antagonistas & inibidores , Fatores de Elongação da Transcrição/biossíntese , Fatores de Elongação da Transcrição/genética , Regulação para Cima/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Hepatology ; 69(6): 2502-2517, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30723918

RESUMO

Hepatocellular carcinoma (HCC) cells exploit an aberrant transcriptional program to sustain their infinite growth and progression. Emerging evidence indicates that the continuous and robust transcription of oncogenes in cancer cells is often driven by super-enhancers (SEs). In this study, we systematically compared the SE landscapes between normal liver and HCC cells and revealed that the cis-acting SE landscape was extensively reprogrammed during liver carcinogenesis. HCC cells acquired SEs at multiple prominent oncogenes to drive their vigorous expression. We identified sphingosine kinase 1 (SPHK1) as an SE-associated oncogene, and we used this gene as an example to illustrate the impact of SEs on the activation of oncogenes in HCC. Concurrently, we also showed that the critical components of the trans-acting SE complex, namely, cyclin-dependent kinase 7 (CDK7), bromodomain-containing protein 4 (BRD4), E1A binding protein P300 (EP300), and mediator complex subunit 1 (MED1), were frequently overexpressed in human HCCs and were associated with the poor prognosis of patients with HCC. Using the CRISPR/Cas9 gene-editing system and specific small-molecule inhibitors, we further demonstrated that HCC cells were highly sensitive to perturbations of the SE complex. The inactivation of CDK7, BRD4, EP300, and MED1 selectively repressed the expression of SE-associated oncogenes in HCC. Finally, we demonstrated that THZ1, which is a small-molecule inhibitor of CDK7, exerted a prominent anticancer effect in both in vitro and in vivo HCC models. Conclusion: The SE landscape and machinery were significantly altered in human HCCs. HCC cells are highly susceptible to perturbations of the SE complex due to the resulting selective suppression of SE-associated oncogenes. Our results suggest that targeting SE complex is a promising therapeutic strategy for HCC treatment.


Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Distribuição de Qui-Quadrado , Proteína p300 Associada a E1A/genética , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Prognóstico , RNA Mensageiro/genética , Medição de Risco , Estatísticas não Paramétricas , Análise de Sobrevida , Fatores de Transcrição/genética , Pesquisa Translacional Biomédica , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/fisiologia
5.
Hepatology ; 67(6): 2254-2270, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29171881

RESUMO

Epigenetic alterations have contributed greatly to human carcinogenesis. Conventional epigenetic studies have predominantly focused on DNA methylation, histone modifications, and chromatin remodeling. Recently, diverse and reversible chemical modifications of RNAs have emerged as a new layer of epigenetic regulation. N6-methyladenosine (m6A) is the most abundant chemical modification of eukaryotic messenger RNA (mRNA) and is important for the regulation of mRNA stability, splicing, and translation. Using transcriptome sequencing, we discovered that methyltransferase-like 3 (METTL3), a major RNA N6-adenosine methyltransferase, was significantly up-regulated in human hepatocellular carcinoma (HCC) and multiple solid tumors. Clinically, overexpression of METTL3 is associated with poor prognosis of patients with HCC. Functionally, we proved that knockdown of METTL3 drastically reduced HCC cell proliferation, migration, and colony formation in vitro. Knockout of METTL3 remarkably suppressed HCC tumorigenicity and lung metastasis in vivo. On the other hand, using the CRISPR/dCas9-VP64 activation system, we demonstrated that overexpression of METTL3 significantly promoted HCC growth both in vitro and in vivo. Through transcriptome sequencing, m6A sequencing, and m6A methylated RNA immuno-precipitation quantitative reverse-transcription polymerase chain reaction, we identified suppressor of cytokine signaling 2 (SOCS2) as a target of METTL3-mediated m6A modification. Knockdown of METTL3 substantially abolished SOCS2 mRNA m6A modification and augmented SOCS2 mRNA expression. We also showed that m6A-mediated SOCS2 mRNA degradation relied on the m6A reader protein YTHDF2-dependent pathway. CONCLUSION: METTL3 is frequently up-regulated in human HCC and contributes to HCC progression. METTL3 represses SOCS2 expression in HCC through an m6A-YTHDF2-dependent mechanism. Our findings suggest an important mechanism of epigenetic alteration in liver carcinogenesis. (Hepatology 2018;67:2254-2270).


Assuntos
Carcinoma Hepatocelular/etiologia , Neoplasias Hepáticas/etiologia , Metiltransferases/fisiologia , Interferência de RNA , Proteínas de Ligação a RNA/fisiologia , Proteínas Supressoras da Sinalização de Citocina/genética , Animais , Carcinoma Hepatocelular/enzimologia , Progressão da Doença , Humanos , Neoplasias Hepáticas/enzimologia , Camundongos
6.
J Hepatol ; 67(4): 758-769, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28532996

RESUMO

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a major leading cause of cancer mortality worldwide. Epigenetic deregulation is a common trait of human HCC. G9s is an important epigenetics regulator however, its role in liver carcinogenesis remains to be investigated. METHODS: Gene expressions were determined by RNA-Seq and qRT-PCR. G9a knockdown and knockout cell lines were established by lentiviral-based shRNA and CRISPR/Cas9 gene editing system. Tumor-promoting functions of G9a was studied in both HCC cell lines and nude mice model. The downstream targets of G9a were identified by RNA-Seq and confirmed by ChIP assay. The therapeutic value of G9a inhibitors was evaluated both in vitro and in vivo. RESULTS: We identified G9a as a frequently upregulated histone methyltransferase in human HCCs. Upregulation of G9a was significantly associated with HCC progression and aggressive clinicopathological features. Functionally, we demonstrated that inactivation of G9a by RNAi knockdown, CRISPR/Cas9 knockout, and pharmacological inhibition remarkably abolished H3K9 di-methylation and suppressed HCC cell proliferation and metastasis in both in vitro and in vivo models. Mechanistically, we showed that the frequent upregulation of G9a in human HCCs was attributed to gene copy number gain at chromosome 6p21. In addition, we identified miR-1 as a negative regulator of G9a. Loss of miR-1 relieved the post-transcriptional repression on G9a and contributed to its upregulation in human HCC. Utilizing RNA sequencing, we identified the tumor suppressor RARRES3 as a critical target of G9a. Epigenetic silencing of RARRES3 contributed to the tumor-promoting function of G9a. CONCLUSION: This study shows a frequent deregulation of miR-1/G9a/RARRES3 axis in liver carcinogenesis, highlighting the pathological significance of G9a and its therapeutic potential in HCC treatment. Lay summary: In this study, we identified G9a histone methyltransferase was frequently upregulated in human HCC and contributes to epigenetic silencing of tumor suppressor gene RARRES3 in liver cancer. Targeting G9a may be a novel approach for HCC treatment.


Assuntos
Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Receptores do Ácido Retinoico/genética , Regiões 3' não Traduzidas , Animais , Carcinoma Hepatocelular/etiologia , Linhagem Celular Tumoral , Epigênese Genética , Dosagem de Genes , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Inativação Gênica , Genes Supressores de Tumor , Humanos , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/etiologia , Neoplasias Hepáticas Experimentais/genética , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Regulação para Cima
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