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1.
Oncogene ; 40(16): 2842-2857, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33742119

RESUMO

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.

2.
J Cell Mol Med ; 25(3): 1568-1582, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33410581

RESUMO

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.

3.
Oncogene ; 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33239755

RESUMO

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.

5.
Adv Sci (Weinh) ; 7(20): 2000157, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33101843

RESUMO

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.

6.
Am J Cancer Res ; 10(8): 2446-2463, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32905496

RESUMO

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.

7.
Theranostics ; 10(22): 9984-10000, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32929329

RESUMO

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.

8.
Theranostics ; 10(23): 10823-10837, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32929382

RESUMO

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.

9.
Am J Pathol ; 190(11): 2267-2281, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32805235

RESUMO

Liver fibrosis is an increasing health problem worldwide, for which no effective antifibrosis drugs are available. Although the involvement of aerobic glycolysis in hepatic stellate cell (HSC) activation has been reported, the role of pyruvate kinase M2 (PKM2) in liver fibrogenesis still remains unknown. We examined PKM2 expression and location in liver tissues and primary hepatic cells. The in vitro and in vivo effects of a PKM2 antagonist (shikonin) and its allosteric agent (TEPP-46) on liver fibrosis were investigated in HSCs and liver fibrosis mouse model. Chromatin immunoprecipitation sequencing and immunoprecipitation were performed to identify the relevant molecular mechanisms. PKM2 expression was significantly up-regulated in both mouse and human fibrotic livers compared with normal livers, and mainly detected in activated, rather than quiescent, HSCs. PKM2 knockdown markedly inhibited the activation and proliferation of HSCs in vitro. Interestingly, the PKM2 dimer, rather than the tetramer, induced HSC activation. PKM2 tetramerization induced by TEPP-46 effectively inhibited HSC activation, reduced aerobic glycolysis, and decreased MYC and CCND1 expression via regulating histone H3K9 acetylation in activated HSCs. TEPP-46 and shikonin dramatically attenuated liver fibrosis in vivo. Our findings demonstrate a nonmetabolic role of PKM2 in liver fibrosis. PKM2 tetramerization or suppression could prevent HSC activation and protects against liver fibrosis.


Assuntos
Células Estreladas do Fígado/enzimologia , Cirrose Hepática/enzimologia , Multimerização Proteica , Piruvato Quinase/metabolismo , Acetilação , Animais , Ciclina D1/metabolismo , Feminino , Células Estreladas do Fígado/patologia , Histonas/metabolismo , Humanos , Cirrose Hepática/patologia , Masculino , Camundongos , Compostos Orgânicos/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo
10.
RNA Biol ; : 1-13, 2020 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-32746693

RESUMO

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.

11.
Signal Transduct Target Ther ; 5(1): 80, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32576812

RESUMO

Human single-stranded DNA-binding protein 1 (hSSB1) is required for the efficient recruitment of the MRN complex to DNA double-strand breaks and is essential for the maintenance of genome integrity. However, the mechanism by which hSSB1 recruits NBS1 remains elusive. Here, we determined that hSSB1 undergoes SUMOylation at both K79 and K94 under normal conditions and that this modification is dramatically enhanced in response to DNA damage. SUMOylation of hSSB1, which is specifically fine-tuned by PIAS2α, and SENP2, not only stabilizes the protein but also enhances the recruitment of NBS1 to DNA damage sites. Cells with defective hSSB1 SUMOylation are sensitive to ionizing radiation, and global inhibition of SUMOylation by either knocking out UBC9 or adding SUMOylation inhibitors significantly enhances the sensitivity of cancer cells to etoposide. Our findings reveal that SUMOylation, as a novel posttranslational modification of hSSB1, is critical for the functions of this protein, indicating that the use of SUMOylation inhibitors (e.g., 2-D08 and ML-792) may be a new strategy that would benefit cancer patients being treated with chemo- or radiotherapy.

12.
13.
Nucleic Acids Res ; 48(D1): D307-D313, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31598693

RESUMO

RNA binding proteins (RBPs) are a large protein family that plays important roles at almost all levels of gene regulation through interacting with RNAs, and contributes to numerous biological processes. However, the complete list of eukaryotic RBPs including human is still unavailable. Here, we systematically identified RBPs in 162 eukaryotic species based on both computational analysis of RNA binding domains (RBDs) and large-scale RNA binding proteomic data, and established a comprehensive eukaryotic RBP database, EuRBPDB (http://EuRBPDB.syshospital.org). We identified a total of 311 571 RBPs with RBDs (corresponding to 6368 ortholog groups) and 3,651 non-canonical RBPs without known RBDs. EuRBPDB provides detailed annotations for each RBP, including basic information and functional annotation. Moreover, we systematically investigated RBPs in the context of cancer biology based on published literatures, PPI-network and large-scale omics data. To facilitate the exploration of the clinical relevance of RBPs, we additionally designed a cancer web interface to systematically and interactively display the biological features of RBPs in various types of cancers. EuRBPDB has a user-friendly web interface with browse and search functions, as well as data downloading function. We expect that EuRBPDB will be a widely-used resource and platform for both the communities of RNA biology and cancer biology.


Assuntos
Neoplasias , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Bases de Dados de Proteínas , Eucariotos , Humanos , Internet , Mutação , Neoplasias/química , Motivos de Ligação ao RNA , Proteínas de Ligação a RNA/genética
14.
Cell Death Dis ; 10(12): 936, 2019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31819048

RESUMO

Chemoresistance is a major cause of cancer progression and the mortality of cancer patients. Developing a safe strategy for enhancing chemosensitivity is a challenge for biomedical science. Recent studies have suggested that vitamin D supplementation may decrease the risk of many cancers. However, the role of vitamin D in chemotherapy remains unknown. We found that vitamin D sensitised oral cancer cells to cisplatin and partially reversed cisplatin resistance. Using RNA-seq, we discovered that lipocalin 2 (LCN2) is an important mediator. Cisplatin enhanced the expression of LCN2 by decreasing methylation at the promoter, whereas vitamin D enhanced methylation and thereby inhibited the expression of LCN2. Overexpression of LCN2 increased cell survival and cisplatin resistance both in vitro and in vivo. High LCN2 expression was positively associated with differentiation, lymph node metastasis, and T staging and predicted a poor prognosis in oral squamous cell carcinoma (OSCC) patients. LCN2 was also associated with post-chemotherapy recurrence. Moreover, we found that LCN2 promoted the activation of NF-κB by binding to ribosomal protein S3 (RPS3) and enhanced the interaction between RPS3 and p65. Our study reveals that vitamin D can enhance cisplatin chemotherapy and suggests that vitamin D should be supplied during chemotherapy; however, more follow-up clinical studies are needed.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/dietoterapia , Cisplatino/farmacologia , Suplementos Nutricionais , Lipocalina-2/metabolismo , Neoplasias Bucais/dietoterapia , NF-kappa B/metabolismo , Proteínas Ribossômicas/metabolismo , Vitamina D/uso terapêutico , Adulto , Animais , Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Seguimentos , Humanos , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Receptores de Calcitriol/genética , Proteínas Ribossômicas/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Carga Tumoral/efeitos dos fármacos , Vitamina D/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
J Cancer ; 10(27): 6837-6847, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31839818

RESUMO

Regulator of chromosome condensation 2 (RCC2), also known as TD-60, is an RCC1 family member and plays an essential role in mitosis. However, the roles of RCC2 in breast cancer are still unclear. In this study, RCC2 was found to exert oncogenic activities in breast cancer. Samples of breast cancer tissue revealed an increased level of RCC2 and a high level of RCC2 was associated with poor overall survival rate of breast cancer patients. Overexpression of RCC2 significantly enhanced cell proliferation and migration abilities of breast cancer cells in vitro and in vivo. Mechanistically, RCC2 induced epithelial-mesenchymal transition (EMT) through the activation of Wnt signaling pathway. Collectively, our study indicates that RCC2 contributes to breast cancer progression and functions as an important regulator of EMT through the activation of Wnt signaling.

16.
EMBO Rep ; 20(5)2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30940648

RESUMO

The bromodomain-containing protein 7 (BRD7) is a tumour suppressor protein with critical roles in cell cycle transition and transcriptional regulation. Whether BRD7 is regulated by post-translational modifications remains poorly understood. Here, we find that chemotherapy-induced DNA damage leads to the rapid degradation of BRD7 in various cancer cell lines. PARP-1 binds and poly(ADP)ribosylates BRD7, which enhances its ubiquitination and degradation through the PAR-binding E3 ubiquitin ligase RNF146. Moreover, the PARP1 inhibitor Olaparib significantly enhances the sensitivity of BRD7-positive cancer cells to chemotherapeutic drugs, while it has little effect on cells with low BRD7 expression. Taken together, our findings show that PARP1 induces the degradation of BRD7 resulting in cancer cell resistance to DNA-damaging agents. BRD7 might thus serve as potential biomarker in clinical trial for the prediction of synergistic effects between chemotherapeutic drugs and PARP inhibitors.


Assuntos
Antineoplásicos/farmacologia , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação/efeitos dos fármacos , Células A549 , Linhagem Celular , Linhagem Celular Tumoral , DNA/metabolismo , Reparo do DNA/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacos
17.
Mob DNA ; 10: 2, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30636978

RESUMO

[This corrects the article DOI: 10.1186/s13100-018-0139-y.].

18.
Nucleic Acids Res ; 47(3): 1255-1267, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30496486

RESUMO

As the second most common malignant bone tumor in children and adolescents, Ewing sarcoma is initiated and exacerbated by a chimeric oncoprotein, most commonly, EWS-FLI1. In this study, we apply epigenomic analysis to characterize the transcription dysregulation in this cancer, focusing on the investigation of super-enhancer and its associated transcriptional regulatory mechanisms. We demonstrate that super-enhancer-associated transcripts are significantly enriched in EWS-FLI1 target genes, contribute to the aberrant transcriptional network of the disease, and mediate the exceptional sensitivity of Ewing sarcoma to transcriptional inhibition. Through integrative analysis, we identify MEIS1 as a super-enhancer-driven oncogene, which co-operates with EWS-FLI1 in transcriptional regulation, and plays a key pro-survival role in Ewing sarcoma. Moreover, APCDD1, another super-enhancer-associated gene, acting as a downstream target of both MEIS1 and EWS-FLI1, is also characterized as a novel tumor-promoting factor in this malignancy. These data delineate super-enhancer-mediated transcriptional deregulation in Ewing sarcoma, and uncover numerous candidate oncogenes which can be exploited for further understanding of the molecular pathogenesis for this disease.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Proteína Meis1/genética , Sarcoma de Ewing/genética , Transcrição Genética , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Humanos , Motivos de Nucleotídeos/genética , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/patologia , Transdução de Sinais/genética
19.
Mob DNA ; 9: 33, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30534207

RESUMO

Background: Similar to retro-/lenti- virus system, DNA transposons are useful tools for stable expression of exogenous genes in mammalian cells. Sleeping Beauty (SB) transposon has adopted for integrating genes into host genomes in recent studies. However, SB-derived vector system for proteins purifying/tracking and gene knockout are still not available. Results: In this study, we generated a series of vectors (termed as pSB vectors) containing Sleeping Beauty IRDR-L/R that can be transposed by SB transposase. Gateway cassette was combined to the pSB vectors to facilitate the cloning. Vectors with various tags, Flag, Myc, HA, V5 and SFB, were generated for multiple options. Moreover, we incorporated the CRISPR-Cas9 cassette into the pSB plasmids for gene knockout. Indeed, using one of these vectors (pSB-SFB-GFP), we performed Tandem Affinity Purification and identified that NFATc1 is a novel binding partner of FBW7. We also knocked out RCC2 and BRD7 using pSB-CRISPR vector respectively, and revealed the novel roles of these two proteins in mitosis. Conclusion: Our study demonstrated that the pSB series vectors are convenient and powerful tools for gene overexpression and knockout in mammalian cells, providing a new alternative approach for molecular cell biology research.

20.
Theranostics ; 8(5): 1286-1300, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29507620

RESUMO

Rationale: Mxi1 is regarded as a potential tumor suppressor protein that antagonizes the transcriptional activity of proto-oncogene Myc. However, the clinical significances and underlying mechanisms by which Mxi1 is regulated in lung cancer remain poorly understood. Methods: Mass spectrometry analysis and immunoprecipitation assay were utilized to detect the protein-protein interaction. The phosphorylation of Mxi1 was evaluated by in vitro kinase assays. Poly-ubiquitination of Mxi1 was examined by in vivo ubiquitination assay. Lung cancer cells stably expressing wild-type Mxi1 or Mxi1-S160A were used for functional analyses. The expression levels of Mxi1 and S6K1 were determined by immunohistochemistry in lung cancer tissues and adjacent normal lung tissues. Results: We found that Mxi1 is downregulated and correlated with poor prognosis in lung cancer. Using tandem affinity purification technology, we provided evidence that ß-Trcp E3 ubiquitin ligase interacts with and promotes the ubiquitination and degradation of Mxi1. Furthermore, we demonstrated that Mxi1 is phosphorylated at S160 site by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase ß-Trcp. Moreover, a phosphorylation mutant form of Mxi1 (Mxi1-S160A), which cannot be degraded by S6K1 and ß-Trcp, is much more stable and efficient in suppressing the transcriptional activity of Myc and radioresistance in lung cancer cells. More importantly, a strong inverse correlation between S6K1 and Mxi1 expression was observed in human lung cancer tissues. Conclusion: Our findings not only establish a crosstalk between the mTOR/S6K1 signaling pathway and Myc activation, but also suggest that targeting S6K1/Mxi1 pathway is a promising therapeutic strategy for the treatment of lung cancer.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Pulmonares/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-myc/metabolismo , Tolerância a Radiação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Neoplasias Pulmonares/patologia , Fosforilação , Fosfosserina/metabolismo , Prognóstico , Ligação Proteica , Estabilidade Proteica , Proteínas Supressoras de Tumor/química , Ubiquitina/metabolismo
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