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1.
Zhonghua Gan Zang Bing Za Zhi ; 28(10): 868-875, 2020 Oct 20.
Artigo em Chinês | MEDLINE | ID: mdl-33105933

RESUMO

Objective: To construct RNA interference (RNAi) lentiviral expression vector of DEK gene, and to explore its effect on the biological behavior of liver cancer cells. Methods: Double-stranded oligo DNAs were annealed and synthesized according to the interference sequence of DEK gene by RNAi technology. Small interfering RNA expression vector pLKO.1 was cloned after enzymatic digestion. The recombinant lentiviral pLKO.1-sh hDEK was constructed, and then the virus supernatant was collected, packed and infected by 293T cells. Real-time reverse transcription PCR (RT-PCR) and Western blot were used to detect DEK expression in human liver cancer cells Bel-7402, Hu-7, SmMC-7721 and HepG2, and DEK knockdown efficiency in each group of lentivirus-infected cells. Cell proliferation ability, cloning ability, apoptosis and migration ability were detected by cell counting kit-8 (CCK8), flow cytometry and scratch test, respectively. The t-test was used to compare the mean between the two groups, and one-way ANOVA was used to compare the multiple groups. Results: Enzymatic digestion and DNA sequencing results confirmed that the recombinant lentiviral vectors pLKO.1-sh hDEK1 and pLKO.1-sh hDEK3 were successfully constructed. RT-PCR and Western blot results showed that the expression of DEK in human liver cancer cells BEL-7402 and Huh7 cells was higher, and pLKO.1-sh hDEK3 was more effective in inhibiting the DEK gene expression (P < 0.05). Therefore, pLKO.1-sh hDEK3 was selected to infect BEL-7402 and Huh7 cells for subsequent functional experiments. CCK8 cell proliferation test result showed that the cell proliferation ability of BEL-7402 and Huh7 cells infected with recombinant lentivirus was weakened when compared with blank control and negative control group (P < 0.05). Apoptosis results showed that the apoptosis rate of knockdown group was higher than that of blank and the negative control group (P < 0.05). Cell scratch test result showed that the wound healing rate of knockdown group was lower than that of blank control and negative control group (P < 0.05), and the difference was statistically significant; however, there was no statistically significant difference between blank control and negative control group. Conclusion: Targeting DEK expression in silent liver cancer cells can inhibit the cell proliferation, migration ability, and induce apoptosis, which lays the foundation for further study of the role of DEK gene in liver cancer.


Assuntos
Proteínas Cromossômicas não Histona/genética , Vetores Genéticos , Neoplasias Hepáticas , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Interferência de RNA , Linhagem Celular Tumoral , Humanos , Lentivirus/genética , Neoplasias Hepáticas/genética , Transfecção
2.
Nat Commun ; 11(1): 4676, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938922

RESUMO

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner.


Assuntos
Fator 4 Ativador da Transcrição/genética , Fatores de Iniciação em Eucariotos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Códon , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Neoplasias/genética , Proteínas Oncogênicas/genética , Oncogenes , Fases de Leitura Aberta , RNA Mensageiro , RNA de Transferência/genética , RNA de Transferência/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Ribossomos/genética
3.
Cell Prolif ; 53(10): e12908, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32951278

RESUMO

OBJECTIVES: In this study, we comprehensively analysed the role of ubiquitin-specific protease 1(USP1) in hepatocellular carcinoma (HCC) using data from a set of public databases. MATERIALS AND METHODS: We analysed the mRNA expression of USP1 in HCC and subgroups of HCC using Oncomine and UALCAN. Survival analysis of USP1 in HCC was conducted with the Kaplan-Meier Plotter database. The mutations of USP1 in HCC were analysed using cBioPortal and the Catalogue of Somatic Mutations in Cancer database. Differential genes correlated with USP1 and WD repeat domain 48 (WDR48) were obtained using LinkedOmics. USP1 was knocked down with small interfering RNA (siRNA) or pharmacologically inhibited by ML-323 in MHCC97H or SK-Hep-1 cell lines for function analysis. RESULTS: High USP1 expression predicted unfavourable overall survival in HCC patients. USP1 showed positive correlations with the abundances of macrophages and neutrophils. We identified 98 differential genes that were positively correlated with both USP1 and WDR48. These genes were mainly involved in the cell cycle, aldosterone synthesis and secretion and oestrogen signalling pathways. Interactions between USP1 and WDR 48 were confirmed using co-immunoprecipitation. USP1 knockdown or ML-323 treatment reduced the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E1. CONCLUSIONS: Overall, USP1 is a promising target for HCC treatment with good prognostic value. USP1 and WDR48 function together in regulating cancer cell proliferation via the cell cycle.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteases Específicas de Ubiquitina/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Intervalo Livre de Doença , Inibidores Enzimáticos/farmacologia , Estrogênios/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Prognóstico , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Mapas de Interação de Proteínas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/genética
4.
Proc Natl Acad Sci U S A ; 117(34): 20776-20784, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788348

RESUMO

Transcription factor fusions (TFFs) are present in ∼30% of soft-tissue sarcomas. TFFs are not readily "druggable" in a direct pharmacologic manner and thus have proven difficult to target in the clinic. A prime example is the CIC-DUX4 oncoprotein, which fuses Capicua (CIC) to the double homeobox 4 gene, DUX4. CIC-DUX4 sarcoma is a highly aggressive and lethal subtype of small round cell sarcoma found predominantly in adolescents and young adults. To identify new therapeutic targets in CIC-DUX4 sarcoma, we performed chromatin immunoprecipitation sequencing analysis using patient-derived CIC-DUX4 cells. We uncovered multiple CIC-DUX4 targets that negatively regulate MAPK-ERK signaling. Mechanistically, CIC-DUX4 transcriptionally up-regulates these negative regulators of MAPK to dampen ERK activity, leading to sustained CIC-DUX4 expression. Genetic and pharmacologic MAPK-ERK activation through DUSP6 inhibition leads to CIC-DUX4 degradation and apoptotic induction. Collectively, we reveal a mechanism-based approach to therapeutically degrade the CIC-DUX4 oncoprotein and provide a precision-based strategy to combat this lethal cancer.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Sarcoma/metabolismo , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Feminino , Genes Homeobox , Humanos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos SCID , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Oncogênicas/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Repressoras/genética , Sarcoma/genética , Sarcoma de Ewing/genética , Sarcoma de Células Pequenas/genética , Fatores de Transcrição/genética , Translocação Genética/genética
5.
Int J Clin Oncol ; 25(9): 1563-1569, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32656741

RESUMO

DEK is a highly conserved nuclear factor that plays an important role in the regulation of multiple cellular processes. DEK was discovered to be an oncogene as a fusion with NUP214 gene, which results in producing DEK-NUP214 proteins, in a subset of patients with acute myeloid leukemia. Subsequently, DEK overexpression was reported in many cancers, thus DEK itself is considered to be an oncoprotein. DEK has been reported to play important roles in the progression of early and late stage squamous cell carcinoma (SCC) and is useful for early diagnosis of the disease. These findings have made DEK an attractive therapeutic target, especially for human papillomavirus (HPV)-associated SCC. However, the mechanism of DEK in SCC remains unclear. In this review, we discuss human DEK oncogene-related SCC.


Assuntos
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Proteínas Cromossômicas não Histona/genética , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Carcinoma de Células Escamosas/diagnóstico , Regulação Neoplásica da Expressão Gênica , Humanos
6.
Nat Commun ; 11(1): 3013, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541654

RESUMO

B lymphoid development is initiated by the differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating mature B cells. This highly regulated process generates clonal immunological diversity via recombination of immunoglobulin V, D and J gene segments. While several transcription factors that control B cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for early B lymphoid differentiation. Erg initiates a transcriptional network involving the B cell lineage defining genes, Ebf1 and Pax5, which directly promotes expression of key genes involved in V(D)J recombination and formation of the B cell receptor. Complementation of Erg deficiency with a productively rearranged immunoglobulin gene rescued B lineage development, demonstrating that Erg is an essential and stage-specific regulator of the gene regulatory network controlling B lymphopoiesis.


Assuntos
Linfócitos B/metabolismo , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Linfopoese/genética , Proteínas Oncogênicas/genética , Transcrição Genética , Regulador Transcricional ERG/genética , Animais , Linfócitos B/citologia , Linhagem da Célula/genética , Células Cultivadas , Redes Reguladoras de Genes/genética , Células-Tronco Hematopoéticas/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Oncogênicas/metabolismo , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulador Transcricional ERG/metabolismo , Recombinação V(D)J/genética
7.
J Biomed Sci ; 27(1): 75, 2020 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576196

RESUMO

BACKGROUND: ZNF322A is an oncogenic transcription factor that belongs to the Cys2His2-type zinc-finger protein family. Accumulating evidence suggests that ZNF322A may contribute to the tumorigenesis of lung cancer, however, the ZNF322A-mediated downstream signaling pathways remain unknown. METHODS: To uncover ZNF322A-mediated functional network, we applied phosphopeptide enrichment and isobaric labeling strategies with mass spectrometry-based proteomics using A549 lung cancer cells, and analyzed the differentially expressed proteins of phosphoproteomic and proteomic profiles to determine ZNF322A-modulated pathways. RESULTS: ZNF322A highlighted a previously unidentified insulin signaling, heat stress, and signal attenuation at the post-translational level. Consistently, protein-phosphoprotein-kinase interaction network analysis revealed phosphorylation of IRS1 and HSP27 were altered upon ZNF322A-silenced lung cancer cells. Thus, we further investigated the molecular regulation of ZNF322A, and found the inhibitory transcriptional regulation of ZNF322A on PIM3, which was able to phosphorylate IRS1 at serine1101 in order to manipulate glucose uptake via the PI3K/AKT/mTOR signaling pathway. Moreover, ZNF322A also affects the unfolded protein response by phosphorylation of HSP27S82 and eIF2aS51, and triggers autophagosome formation in lung cancer cells. CONCLUSIONS: These findings not only give new information about the molecular regulation of the cellular proteins through ZNF322A at the post-translational level, but also provides a resource for the study of lung cancer therapy.


Assuntos
Autofagossomos/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Neoplasias Pulmonares/genética , Proteínas Oncogênicas/genética , Fatores de Transcrição/genética , Resposta a Proteínas não Dobradas , Células A549 , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Oncogênicas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismo
8.
PLoS Pathog ; 16(4): e1008541, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32353058

RESUMO

Ehrlichia chaffeensis (E. chaffeensis) exploits evolutionarily conserved Notch and Wnt host cell signaling pathways to downregulate innate immune host defenses and promote infection. The multifunctional E. chaffeensis TRP120 effector which has HECT E3 ubiquitin ligase activity, interacts with the host nuclear tumor suppressor F-BOX and WD domain repeating-containing 7 (FBW7). FBW7 is the substrate recognition subunit of the Skp1-cullin-1-FBOX E3 ubiquitin (Ub) ligase complex (SCF) known to negatively regulate a network of oncoproteins (Notch, cyclin E, c-Jun, MCL1 and cMYC). In this study, we demonstrate that TRP120 and FBW7 colocalize strongly in the nucleus by confocal immunofluorescent microscopy and interactions between TRP120 and FBW7 FBOX and WD40 domains were demonstrated by ectopic expression and co-immunoprecipitation. Although FBW7 gene expression increased during E. chaffeensis infection, FBW7 levels significantly decreased (>70%) by 72 h post infection. Moreover, an iRNA knockdown of FBW7 coincided with increased E. chaffeensis infection and levels of Notch intracellular domain (NICD), phosphorylated c-Jun, MCL-1 and cMYC, which are negatively regulated by FBW7. An increase in FBW7 K48 ubiquitination was detected during infection by co-IP, and FBW7 degradation was inhibited in infected cells treated with the proteasomal inhibitor bortezomib. Direct TRP120 ubiquitination of native and recombinant FBW7 was demonstrated in vitro and confirmed by ectopic expression of TRP120 HECT Ub ligase catalytic site mutant. This study identifies the tumor suppressor, FBW7, as a TRP120 HECT E3 Ub ligase substrate, and demonstrates that TRP120 ligase activity promotes ehrlichial infection by degrading FBW7 to maintain stability of Notch and other oncoproteins involved in cell survival and apoptosis.


Assuntos
Ehrlichia chaffeensis/metabolismo , Ehrlichiose/genética , Proteína 7 com Repetições F-Box-WD/metabolismo , Apoptose/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ehrlichia chaffeensis/genética , Ehrlichia chaffeensis/fisiologia , Ehrlichiose/metabolismo , Proteínas F-Box/metabolismo , Proteína 7 com Repetições F-Box-WD/genética , Interações Hospedeiro-Patógeno , Humanos , Proteínas Oncogênicas/genética , Ligação Proteica/fisiologia , Células THP-1 , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
9.
Am J Surg Pathol ; 44(9): 1244-1250, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32366754

RESUMO

Primary squamous cell carcinomas (SCCs) of the middle ear and temporal bone are rare and usually keratinizing by morphology. Nonkeratinizing, basaloid SCCs arising in this area are exceedingly rare, and, due to the anatomic proximity to the skull base, nasopharynx, and nasal sinuses, the differential diagnosis is broad. Most tumors with squamous differentiation arising in these subsites are either viral-induced (human papillomavirus/Epstein-Barr virus) or rarely may have specific molecular alterations (BRD4-NUT, EWSR1-FLI translocations). Occasional tumors are negative for these findings, and their pathogenesis is unknown. A recently discovered DEK-AFF2 fusion was clinically detected in a series of 2 cases known to the authors. This fusion has been previously reported in the literature in a patient with a base of skull tumor who was an exceptional responder to programmed cell death protein 1 inhibitor therapy. We examine here the histomorphologic and molecular findings of 2 additional cases of an emerging entity. Two male patients were identified. Each had a primary middle ear/temporal bone mass with locally advanced disease. The histology was reviewed, and immunohistochemistry was performed. RNA-based next-generation sequencing was performed for clinical detection of diagnostic or actionable fusions. Both patients had basaloid/nonkeratinizing tumors on biopsy. They were positive for markers of squamous differentiation (HMWK, CK5, and p40). By RNA sequencing, they demonstrated the presence of a DEK-AFF2 fusion and were negative for EWSR1 and NUT translocations. The DEK-AFF2 fusion may define a novel diagnostic category of middle ear and temporal bone nonkeratinizing/basaloid SCCs. This fusion also may represent a potential avenue for immunotherapy in these patients. Further studies are needed to fully explore whether this fusion defines a location-specific clinicopathologic entity.


Assuntos
Biomarcadores Tumorais/genética , Proteínas Cromossômicas não Histona/genética , Neoplasias da Orelha/genética , Orelha Média , Fusão Gênica , Neoplasias de Cabeça e Pescoço/genética , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Neoplasias Cranianas/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Osso Temporal , Neoplasias da Orelha/patologia , Neoplasias da Orelha/cirurgia , Orelha Média/patologia , Orelha Média/cirurgia , Predisposição Genética para Doença , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Neoplasias Cranianas/patologia , Neoplasias Cranianas/cirurgia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/cirurgia , Osso Temporal/patologia , Osso Temporal/cirurgia
10.
Nat Microbiol ; 5(8): 1051-1063, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32424339

RESUMO

To accomplish the remarkable task of lifelong infection, the Epstein-Barr virus (EBV) switches between four viral genome latency and lytic programmes to navigate the B-cell compartment and evade immune responses. The transforming programme, consisting of highly immunogenic EBV nuclear antigen (EBNA) and latent membrane proteins (LMPs), is expressed in newly infected B lymphocytes and in post-transplant lymphomas. On memory cell differentiation and in most EBV-associated Burkitt's lymphomas, all but one viral antigen are repressed for immunoevasion. To gain insights into the epigenetic mechanisms that restrict immunogenic oncoprotein expression, a genome-scale CRISPR-Cas9 screen was performed in EBV and Burkitt's lymphoma cells. Here, we show that the ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) and its DNA methyltransferase partner DNA methyltransferase I (DNMT1) are critical for the restriction of EBNA and LMP expression. All UHRF1 reader and writer domains were necessary for silencing and DNMT3B was identified as an upstream viral genome CpG methylation initiator. Polycomb repressive complex I exerted a further layer of control over LMP expression, suggesting a second mechanism for latency programme switching. UHRF1, DNMT1 and DNMT3B are upregulated in germinal centre B cells, the Burkitt's lymphoma cell of origin, providing a molecular link between B-cell state and the EBV latency programme. These results suggest rational therapeutic targets to manipulate EBV oncoprotein expression.


Assuntos
Linfócitos B/virologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/farmacologia , Metilação de DNA/fisiologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 4/efeitos dos fármacos , Herpesvirus Humano 4/genética , Proteínas Oncogênicas/metabolismo , Antígenos Virais , Linfoma de Burkitt , Proteínas Estimuladoras de Ligação a CCAAT , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases , Antígenos Nucleares do Vírus Epstein-Barr , Genes Virais , Genoma Viral , Herpesvirus Humano 4/metabolismo , Humanos , Proteínas Oncogênicas/genética , Ubiquitina-Proteína Ligases
11.
Anticancer Res ; 40(4): 1855-1866, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32234873

RESUMO

BACKGROUND/AIM: The resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib or erlotinib, is considered a major challenge in the treatment of patients with non-small cell lung cancer (NSCLC). Herein, we identified the critical roles of anterior gradient 2 (AGR2) in gefitinib (Gef) resistance of mutant NSCLC cells. MATERIALS AND METHODS: Using datasets from a pair of NSCLC-sensitive and NSCLC-resistant cells, immunoblotting, immunofluorescence and immunohistochemistry, and cell viability assays were applied to identify the effects of AGR2. RESULTS: AGR2 was found to be significantly over-expressed in Gef-resistant cells and was highly associated with drug resistance, proliferation, migration, and invasion of cancer cells. Moreover, AGR2 and ADAMTS6 formed a negative feedback loop in drug-resistant cells. CONCLUSION: Modulation of overexpression of AGR2 in mutant NSCLC cells may be an attractive therapeutic strategy for the treatment of EGFR-TKI-resistant NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Mucoproteínas/genética , Proteínas Oncogênicas/genética , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Cloridrato de Erlotinib/farmacologia , Gefitinibe/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Quinazolinas/farmacologia
12.
Gene ; 744: 144608, 2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-32234541

RESUMO

Prostate cancer (PCa) is the third most common malignancy worldwide. Novel and effective therapeutic targets are needed for PCa. The purpose of this study was to discover novel therapeutic targets for PCa by performing advanced analysis on PCa RNA sequencing (RNAseq) data from The Cancer Genome Atlas (TCGA). Weighted correlation-network analysis (WGCNA) was performed on the RNAseq data of tumor samples, and the module most relevant to the Gleason score was identified. Combining differential gene-expression analysis and survival analysis, we narrowed down potential therapeutic target genes and found that PKMYT1 might be one. Subsequently, functional studies (i.e., cell-proliferation assays, cell cycle analysis, and colony-formation assays) demonstrated that knockdown of PKMYT1 significantly inhibited the growth of PCa cells. Further investigation illustrated that PKMYT1 promoted the growth of PCa cells through targeting CCNB1 and CCNE1 expression. In addition, fostamatinib, an inhibitor of PKMYT1, effectively inhibited the proliferation of PCa cells. Taken together, our results suggest that PKMYT1 is a gene associated with malignancy of PCa and is a novel therapeutic target.


Assuntos
Neoplasias da Próstata/enzimologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Humanos , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Oxazinas/uso terapêutico , Prognóstico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Piridinas/uso terapêutico
13.
Hum Cell ; 33(3): 790-800, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32304027

RESUMO

Anterior gradient 2 (AGR2) was proved to modulate cancer progression. However, the role of AGR2 on endometrial cancer was not established. Here, we investigated the effects of AGR2 expression on endometrial cancer and explored the regulation mechanism. In the study, we found that AGR2 was overexpressed in tumor tissues of 30 endometrial cancer patients. A high level of AGR2 promoted endometrial cancer cells proliferation, migration and invasion. AGR2 induced the expression of lactate dehydrogenase A (LDHA), phosphoglycerate kinase 1 (PGK1), kallikrein 2 (HK2), and enolase 1-α (ENO1), glucose uptake and lactate production. AGR2 could bind to MUC1 and induce MUC1 and hypoxia-inducible factor 1α (HIF-1α). The inhibition effects of AGR2 knockdown on cells proliferation, migration and invasion ability were abolished by the overexpression of MUC1. Besides, the overexpression of MUC1 also reversed the inhibition effects of AGR2 knockdown on the expression of LDHA, HK2, PGK1 and ENO1, glucose uptake and lactate production. AGR2 knockdown inhibited tumor growth, the levels of Ki-67, MUC1, HIF-1α and glycolysis. In conclusion, AGR2 was overexpressed in endometrial cancer and AGR2-induced glucose metabolism facilitated the progression of endometrial carcinoma via the MUC1/HIF-1α pathway. AGR2 may be an effective therapeutic target for endometrial carcinoma.


Assuntos
Carcinoma/genética , Carcinoma/patologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Glucose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mucina-1/genética , Mucina-1/metabolismo , Mucoproteínas/fisiologia , Proteínas Oncogênicas/fisiologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma/metabolismo , Carcinoma/terapia , Movimento Celular/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/terapia , Feminino , Expressão Gênica , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Terapia de Alvo Molecular , Mucoproteínas/genética , Mucoproteínas/metabolismo , Invasividade Neoplásica/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
14.
Nat Struct Mol Biol ; 27(4): 392-399, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251413

RESUMO

The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.


Assuntos
Membrana Celular/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Proteínas Oncogênicas/ultraestrutura , Membrana Celular/química , Membrana Celular/genética , Citocinese/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/química , Endossomos/genética , Endossomos/ultraestrutura , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Polimerização , Conformação Proteica
15.
Biochim Biophys Acta Mol Cell Res ; 1867(8): 118707, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32243901

RESUMO

The gene encoding promyelocytic leukemia protein (PML) generates several spliced isoforms. Ectopic expression of PML1 promotes the proliferation of ERα-positive MCF-7 breast cancer (BC) cells, while a loss of PML by knockdown or overexpression of PML4 does the opposite. PML is an essential constituent of highly dynamic particles called PML nuclear bodies (NBs). PML NBs are heterogenous multiprotein subnuclear structures that are part of cellular stress sensing machinery. The antioxidant sulforaphane (SFN) inhibits the proliferation of BC cells and causes a redistribution of the subcellular localization of PML, a disruption of disulfide-bond linkages in nuclear PML-containing complexes, and a reduction in the number and size of PML NBs. Mechanistically, SFN modifies several cysteine residues, including C204, located in the RBCC domain of PML. PML is sumoylated and contains a Sumo-interacting motif, and a significant fraction of Sumo1 and Sumo2/3 co-localizes with PML NBs. Ectopic expression of the mutant C204A selectively inhibits the biogenesis of endogenous PML NBs but not PML-less Sumo1-, Sumo2/3, or Daxx-containing nuclear speckles. Importantly, PML1 (C204A) functions as a dominant-negative mutant over endogenous PML protein and promotes anti-proliferation activity. Together, we conclude that SFN elicits its cytotoxic activity in part by inactivating PML1's pro-tumorigenic activity.


Assuntos
Antioxidantes/metabolismo , Isotiocianatos/farmacologia , Proteínas Oncogênicas/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Isoformas de Proteínas/metabolismo , Ciclo Celular , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas Correpressoras , Humanos , Células MCF-7 , Chaperonas Moleculares , Proteínas Oncogênicas/genética , Isoformas de Proteínas/genética , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina , Sumoilação , Ubiquitinas/metabolismo
16.
Bull Cancer ; 107(4): 447-457, 2020 Apr.
Artigo em Francês | MEDLINE | ID: mdl-32067719

RESUMO

The advent of molecular biology resulted in the discovery of new oncogenes that have led to the development of targeted therapies for the management of cancer patients. The development of these therapies has improved the prognosis of patients in various tumour localizations. The TRK receptor (tropomyosin receptor kinase) is a transmembrane receptor with a tyrosine kinase activity that plays a role in both cell proliferation and the physiology of the nervous system. Fusions involving the NTRK gene, which codes for this receptor, have been found in different types of solid tumours and lead to its constitutional activation. These fusions, however uncommon, are mainly found in rare pediatric tumours but can also be encountered in digestive cancers with high prevalence (such as colorectal cancer, especially in case of microsatellite instability, with a frequency of 2.5 to 38.5 %) or in aggressive cancers (such as pancreatic cancer). Therapies targeting TRK, such as larotrectinib or entrectinib, have shown significant response rates, usually greater than 6 months, for tumours from various primary sites presenting NTRK fusions and refractory to standard therapies. These fusions can be detected by different methods: immunohistochemistry, FISH (fluorescence in situ hybridization) as well as NGS (next generation sequencing). The intent of this review is to report on current knowledge on NTRK fusions in oncology and to discuss the role of these fusions in digestive cancers and potential therapeutic implications.


Assuntos
Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Fusão Gênica , Fatores de Crescimento Neural/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Oncogênicas/genética , Benzamidas/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Hibridização In Situ , Indazóis/uso terapêutico , Glicoproteínas de Membrana/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Receptor trkA/genética , Receptor trkB/genética , Receptor trkC/genética
17.
Nat Rev Mol Cell Biol ; 21(5): 255-267, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32071436

RESUMO

Oncoproteins of the MYC family are major drivers of human tumorigenesis. Since a large body of evidence indicates that MYC proteins are transcription factors, studying their function has focused on the biology of their target genes. Detailed studies of MYC-dependent changes in RNA levels have provided contrasting models of the oncogenic activity of MYC proteins through either enhancing or repressing the expression of specific target genes, or as global amplifiers of transcription. In this Review, we first summarize the biochemistry of MYC proteins and what is known (or is unclear) about the MYC target genes. We then discuss recent progress in defining the interactomes of MYC and MYCN and how this information affects central concepts of MYC biology, focusing on mechanisms by which MYC proteins modulate transcription. MYC proteins promote transcription termination upon stalling of RNA polymerase II, and we propose that this mechanism enhances the stress resilience of basal transcription. Furthermore, MYC proteins coordinate transcription elongation with DNA replication and cell cycle progression. Finally, we argue that the mechanism by which MYC proteins regulate the transcription machinery is likely to promote tumorigenesis independently of global or relative changes in the expression of their target genes.


Assuntos
Proteína Proto-Oncogênica N-Myc/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Transcrição Genética , Carcinogênese/genética , Ciclo Celular/genética , Proliferação de Células/genética , Replicação do DNA/genética , Humanos , Proteínas Oncogênicas/genética , Fatores de Transcrição
18.
Indian J Pathol Microbiol ; 63(1): 103-105, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32031134

RESUMO

Nuclear protein in testis (NUT) midline carcinoma is poorly differentiated carcinoma defined by rearrangement of NUT gene on 15 to other genes, usually BRD4 on 19. It is first described in 1991. These tumors are most commonly seen in the mediastinum and 35% occur in head and neck. It is a highly aggressive tumor with a median survival of 7 months because of ineffective chemotherapy and undefined treatment. Hence, we must differentiate these tumors from other poorly differentiated tumors. Here, we present a case of NUT midline carcinoma of 44-year male, who presented with headache and dizziness, confirmed by immunohistochemistry of NUT antibody. The aim of this case report is to increase the awareness about this entity in adults with brief review of relevant literature.


Assuntos
Carcinoma/diagnóstico por imagem , Carcinoma/patologia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Neoplasias de Tecidos Moles/diagnóstico por imagem , Seio Esfenoidal/diagnóstico por imagem , Adulto , Carcinoma/classificação , Proteínas de Ciclo Celular/genética , Rearranjo Gênico , Técnicas Histológicas , Humanos , Imuno-Histoquímica , Masculino , Neoplasias de Tecidos Moles/patologia , Seio Esfenoidal/patologia , Tomografia Computadorizada por Raios X , Fatores de Transcrição/genética
19.
Gastroenterology ; 158(6): 1682-1697.e1, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32032585

RESUMO

BACKGROUND & AIMS: Esophageal adenocarcinomas (EACs) are heterogeneous and often preceded by Barrett's esophagus (BE). Many genomic changes have been associated with development of BE and EAC, but little is known about epigenetic alterations. We performed epigenetic analyses of BE and EAC tissues and combined these data with transcriptome and genomic data to identify mechanisms that control gene expression and genome integrity. METHODS: In a retrospective cohort study, we collected tissue samples and clinical data from 150 BE and 285 EAC cases from the Oesophageal Cancer Classification and Molecular Stratification consortium in the United Kingdom. We analyzed methylation profiles of all BE and EAC tissues and assigned them to subgroups using non-negative matrix factorization with k-means clustering. Data from whole-genome sequencing and transcriptome studies were then incorporated; we performed integrative methylation and RNA-sequencing analyses to identify genes that were suppressed with increased methylation in promoter regions. Levels of different immune cell types were computed using single-sample gene set enrichment methods. We derived 8 organoids from 8 EAC tissues and tested their sensitivity to different drugs. RESULTS: BE and EAC samples shared genome-wide methylation features, compared with normal tissues (esophageal, gastric, and duodenum; controls) from the same patients and grouped into 4 subtypes. Subtype 1 was characterized by DNA hypermethylation with a high mutation burden and multiple mutations in genes in cell cycle and receptor tyrosine signaling pathways. Subtype 2 was characterized by a gene expression pattern associated with metabolic processes (ATP synthesis and fatty acid oxidation) and lack methylation at specific binding sites for transcription factors; 83% of samples of this subtype were BE and 17% were EAC. The third subtype did not have changes in methylation pattern, compared with control tissue, but had a gene expression pattern that indicated immune cell infiltration; this tumor type was associated with the shortest time of patient survival. The fourth subtype was characterized by DNA hypomethylation associated with structure rearrangements, copy number alterations, with preferential amplification of CCNE1 (cells with this gene amplification have been reported to be sensitive to CDK2 inhibitors). Organoids with reduced levels of MGMT and CHFR expression were sensitive to temozolomide and taxane drugs. CONCLUSIONS: In a comprehensive integrated analysis of methylation, transcriptome, and genome profiles of more than 400 BE and EAC tissues, along with clinical data, we identified 4 subtypes that were associated with patient outcomes and potential responses to therapy.


Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Metilação de DNA/genética , Epigênese Genética/genética , Mucosa Esofágica/patologia , Neoplasias Esofágicas/genética , Adenocarcinoma/patologia , Idoso , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Esôfago de Barrett/tratamento farmacológico , Esôfago de Barrett/patologia , Ciclina E/genética , Metilação de DNA/efeitos dos fármacos , Progressão da Doença , Epigênese Genética/efeitos dos fármacos , Neoplasias Esofágicas/patologia , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Oncogênicas/genética , Regiões Promotoras Genéticas/genética , RNA-Seq , Estudos Retrospectivos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Sequenciamento Completo do Genoma
20.
PLoS Pathog ; 16(2): e1008105, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32092124

RESUMO

Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins-EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.


Assuntos
Morte Celular Autofágica/efeitos dos fármacos , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Leupeptinas/farmacologia , Lisossomos/metabolismo , Proteínas Oncogênicas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Animais , Antígenos Nucleares do Vírus Epstein-Barr/genética , Células HEK293 , Herpesvirus Humano 4/genética , Humanos , Lisossomos/genética , Camundongos , Proteínas Oncogênicas/genética , Complexo de Endopeptidases do Proteassoma/genética
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