RESUMO
Transcriptional reactivation of hTERT is the limiting step in tumorigenesis. While mutations in hTERT promoter present in 19% of cancers are recognized as key drivers of hTERT reactivation, mechanisms by which wildtype hTERT (WT-hTERT) promoter is reactivated, in majority of human cancers, remain unknown. Using primary colorectal cancers (CRC) we identified Tert INTeracting region 2 (T-INT2), the critical chromatin region essential for reactivating WT-hTERT promoter in CRCs. Elevated ß-catenin and JunD level in CRC facilitates chromatin interaction between hTERT promoter and T-INT2 that is necessary to turn on hTERTexpression. Pharmacological screens uncovered salinomycin, which inhibits JunD mediated hTERT-T-INT2 interaction that is required for the formation of a stable transcription complex on the hTERT promoter. Our results showed for the first time how known CRC alterations, such as APC, lead to WT-hTERT promoter reactivation during stepwise-tumorigenesis and provide a new perspective for developing cancer-specific drugs.
Healthy and cancer cells harbor the same DNA sequence, but reactivation of the Human Telomerase Reverse Transcriptase (hTERT) gene is observed only in cancer cells. How does that happen was not known for over three decades of research? This study identifies a specific DNA structure that forms only in cancer cells and brings the necessary molecular machinery into the correct position to activate the hTERT gene. The detailed mechanism of hTERT activation provided in this study will be instrumental in designing cancer cell-specific hTERT inhibitors, especially since all the other ways of inhibiting telomerase failed in the clinic.
Assuntos
Neoplasias Colorretais , Telomerase , Humanos , Carcinogênese , Cromatina/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Regiões Promotoras Genéticas , Telomerase/antagonistas & inibidores , Telomerase/genética , Transcrição GênicaRESUMO
Cancer-specific hTERT promoter mutations reported in 19% of cancers result in enhanced telomerase activity. Understanding the distinctions between transcriptional regulation of wild-type (WT) and mutant (Mut) hTERT promoters may open up avenues for development of inhibitors which specially block hTERT expression in cancer cells. To comprehensively identify physiological regulators of WT- or Mut-hTERT promoters, we generated several isogenic reporter cells driven by endogenous hTERT loci. Genome-wide CRISPR-Cas9 and small interfering RNA screens using these isogenic reporter lines identified specific regulators of Mut-hTERT promoters. We validate and characterize one of these hits, namely, MED12, a kinase subunit of mediator complex. We demonstrate that MED12 specifically drives expression of hTERT from the Mut-hTERT promoter by mediating long-range chromatin interaction between the proximal Mut-hTERT promoter and T-INT1 distal regulatory region 260 kb upstream. Several hits identified in our screens could serve as potential therapeutic targets, inhibition of which may specifically block Mut-hTERT promoter driven telomerase reactivation in cancers.
Assuntos
Mutação , Regiões Promotoras Genéticas , Telomerase/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Cromatina , Proteínas de Ligação a DNA , Edição de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Complexo Mediador/genética , Complexo Mediador/metabolismo , Neoplasias/genética , Sequências Reguladoras de Ácido Nucleico , Telomerase/metabolismo , Fatores de Transcrição , Transcrição GênicaRESUMO
Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation-dependent enrichment of 5'-tsRNAs. We report the identification of a set of 5'-tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5'-tsRNAs revealed a switch in their association with "effector" RNPs and "target" mRNAs in different cell states. We demonstrate that specific 5'-tsRNAs can preferentially interact with the RNA-binding protein, Igf2bp1, in the RA-induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency-promoting factor, c-Myc, thus providing a mechanistic basis for how 5'-tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5'-tsRNAs in defining distinct cell states.
Assuntos
Diferenciação Celular , MicroRNAs/metabolismo , RNA de Transferência/metabolismo , Animais , Células Cultivadas , Células HCT116 , Humanos , Camundongos , MicroRNAs/genética , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Estabilidade de RNA , RNA de Transferência/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Differentiation of human pluripotent stem cells as embryoid bodies (EBs) has been achieved previously with p38alfa MAPK inhibitors such as SB203580 with moderate efficiency of 10-15%. We synthesized and screened 42 compounds that are 2,4,5-trisubstituted azole analogues of SB203580 for efficient cardiomyocyte differentiation. Our screen identified novel compounds that have similar cardiac differentiation activity as SB203580. However, the cardiac differentiation did not correlate with p38alfa MAPK inhibition, indicating an alternative mechanism in cardiac differentiation. Upon profiling several 2,4,5-trisubstituted azole compounds against a panel of 97 kinases we identified several off targets, among them casein kinases 1 (CK1). The cardiomyogenic activities of SB203580 and its analogues showed a correlation with post mesoderm Wnt/beta-catenin pathway inhibition of CK1 epsilon and delta. These findings united the mechanism of 2,4,5-trisubstituted azole with the current theory of Wnt/beta-catenin regulated pathway of cardiac differentiation. Consequently an efficient cardiomyocyte protocol was developed with Wnt activator CHIR99021 and 2,4,5-trisubstituted azoles to give high yields of 50-70% cardiomyocytes and a 2-fold increase in growth.
Assuntos
Caseína Quinase I/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Piridinas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Linhagem Celular , Desenho de Fármacos , Humanos , Imidazóis/síntese química , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Camundongos , Organogênese/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Piridinas/síntese químicaRESUMO
Over-consumption of iron-rich red meat and hereditary or genetic iron overload are associated with increased risk of colorectal carcinogenesis, yet the mechanistic basis of how metal-mediated signaling leads to oncogenesis remains enigmatic. Using fresh colorectal cancer (CRC) samples we identify Pirin, an iron sensor, that overcomes a rate-limiting step in oncogenesis, by re-activating the dormant human-reverse-transcriptase (hTERT) subunit of telomerase holoenzyme in an iron-(Fe3+)-dependent-manner and thereby drives CRCs. Chemical genetic screens combined with isothermal-dose response fingerprinting and mass-spectrometry identified a small molecule SP2509, that specifically inhibits Pirin-mediated hTERT reactivation in CRCs by competing with iron-(Fe3+) binding. Our findings, first to document how metal ions reactivate telomerase, provide a molecular mechanism for the well-known association between red meat, and increased incidence of CRCs. Small molecules like SP2509 represent a novel modality to target telomerase that acts as driver of 90% human cancers and is yet to be targeted in clinic.
RESUMO
The p38α mitogen-activated protein kinase (MAPK) inhibitor SB203580 had been reported to enhance the cardiomyogenesis of human embryonic stem cells (hESCs). To investigate if tri-substituted imidazole analogues of SB203580 are equally effective inducers for cardiomyogenesis of hESCs, and if there is a correlation between p38α MAPK inhibition and cardiomyogenesis, we designed and synthesized a series of novel tri-substituted imidazoles with a range of p38α MAPK inhibitory activities. Our studies demonstrated that suitably designed analogues of SB203580 can also be inducers of cardiomyogenesis in hESCs and that cell growth is affected by changes in the imidazole structures.
Assuntos
Células-Tronco Embrionárias/citologia , Imidazóis/química , Piridinas/química , Diferenciação Celular/efeitos dos fármacos , Humanos , Imidazóis/metabolismo , Imidazóis/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Miócitos Cardíacos/citologia , Ligação Proteica , Piridinas/metabolismoRESUMO
Here we report a molecular docking-based approach to identify small molecules that can target the ß-catenin (ß-cat)-TCF4 protein-protein interaction (PPI), a key effector complex for nuclear Wnt signaling activity. Specifically, we developed and optimized a computational model of ß-cat using publicly available ß-cat protein crystal structures, and existing ß-cat-TCF4 interaction inhibitors as the training set. Using our computational model to an in silico screen predicted 27 compounds as good binders to ß-cat, of which 3 were identified to be effective against a Wnt-responsive luciferase reporter. In vitro functional validation experiments revealed GB1874 as an inhibitor of the Wnt pathway that targets the ß-cat-TCF4 PPI. GB1874 also affected the proliferation and stemness of Wnt-addicted colorectal cancer (CRC) cells in vitro. Encouragingly, GB1874 inhibited the growth of CRC tumor xenografts in vivo, thus demonstrating its potential for further development into therapeutics against Wnt-associated cancer indications.
RESUMO
BACKGROUND: Overexpression of epidermal growth factor receptor (EGFR), and downstream pathway activation appears to be a common oncogenic driver in the majority of head and neck squamous cell cancers (HNSCCs); yet targeting EGFR for the treatment of HNSCC has met with limited success. Apart from the anti-EGFR antibody cetuximab, no small molecule EGFR/tyrosine kinase inhibitors (TKIs) have progressed to routine clinical use. The aim of this study was to determine factors contributing to the lack of response to TKIs and identify alternative therapeutic vulnerabilities. METHODS: Genomic and transcriptomic sequencing, high-throughput compound screens, overexpression and siRNA knockdown, western blot, in vivo xenograft studies. FINDINGS: We derived three pairs of isogenic gefitinib (TKI)-sensitive and resistant patient-derived HNSCC cell lines. Genomic sequencing of gefitinib-resistant cell lines identified a lack of activating and resistance-associated EGFR mutations. Instead, transcriptomic sequencing showed upregulated EMT gene signature in the gefitinib-resistant cells with a corresponding increase in their migratory phenotype. Additionally, the resistant cell displayed reduced growth rate. Surprisingly, while gefitinib-resistant cells were independent of EGFR for survival, they nonetheless displayed activation of downstream ERK and AKT signalling. High-throughput screening (HTS) of druggable, small molecule libraries revealed that the gefitinib-resistant cells were particularly sensitive to inhibitors of genes involved in cell cycle and mitosis, such as Aurora kinase inhibitors (AKIs), cyclin-dependent kinase (CDK) inhibitors, and microtubule inhibitors. Notably our results showed that in the EGFR inhibited state, Aurora kinases are essential for cell survival. INTERPRETATION: Our study demonstrates that in the absence of activating EGFR mutations, HNSCCs may gain resistance to gefitinib through decreased cell proliferation, which makes them exceptionally vulnerable to cell-cycle inhibitors. FUNDING: Agency for Science, Technology, and Research (A*STAR), National Medical Research Council (NMRC), and the National Institutes of Health (NIH)/National Cancer Institute (NCI).
Assuntos
Aurora Quinases/antagonistas & inibidores , Aurora Quinases/metabolismo , Biomarcadores Tumorais , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Gefitinibe/farmacologia , Mutação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores ErbB/genética , Imunofluorescência , Humanos , Modelos Biológicos , Bibliotecas de Moléculas Pequenas , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Genomics-driven cancer therapeutics has gained prominence in personalized cancer treatment. However, its utility in indications lacking biomarker-driven treatment strategies remains limited. Here we present a "phenotype-driven precision-oncology" approach, based on the notion that biological response to perturbations, chemical or genetic, in ex vivo patient-individualized models can serve as predictive biomarkers for therapeutic response in the clinic. We generated a library of "screenable" patient-derived primary cultures (PDCs) for head and neck squamous cell carcinomas that reproducibly predicted treatment response in matched patient-derived-xenograft models. Importantly, PDCs could guide clinical practice and predict tumour progression in two n = 1 co-clinical trials. Comprehensive "-omics" interrogation of PDCs derived from one of these models revealed YAP1 as a putative biomarker for treatment response and survival in ~24% of oral squamous cell carcinoma. We envision that scaling of the proposed PDC approach could uncover biomarkers for therapeutic stratification and guide real-time therapeutic decisions in the future.Treatment response in patient-derived models may serve as a biomarker for response in the clinic. Here, the authors use paired patient-derived mouse xenografts and patient-derived primary culture models from head and neck squamous cell carcinomas, including metastasis, as models for high-throughput screening of anti-cancer drugs.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Medicina de Precisão/métodos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Biomarcadores Tumorais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Gefitinibe , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos Endogâmicos NOD , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Fenótipo , Fosfoproteínas/genética , Quinazolinas/farmacologia , Fatores de Transcrição , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAPRESUMO
SIGNIFICANCE: Protein tyrosine phosphatases (PTPs) are important enzymes that are involved in the regulation of cellular signaling. Evidence accumulated over the years has indicated that PTPs present exciting opportunities for drug discovery against diseases such as diabetes, cancer, autoimmune diseases, and tuberculosis. However, the highly conserved and partially positive charge of the catalytic sites of PTPs is a major challenge in the development of potent and highly selective PTP inhibitors. RECENT ADVANCES: Here, we examine the strategy of developing bidentate inhibitors for selective inhibition of PTPs. Bidentate inhibitors are small-molecular-weight compounds with the ability to bind to both the active site and a non-conserved secondary phosphate binding site. This secondary phosphate binding site was initially discovered in protein tyrosine phosphatase 1B (PTP1B), and, hence, most of the bidentate inhibitors reported in this review are PTP1B inhibitors. CRITICAL ISSUES: Although bidentate inhibition is a good strategy for developing potent and selective inhibitors, the cell membrane permeability and pharmacokinetic properties of the inhibitors are also important for successful drug development. In this review, we will also summarize the various efforts made toward the development of phosphotyrosine (pTyr) mimetics for increasing cellular permeability. FUTURE DIRECTIONS: Even though the secondary phosphate binding site was initially found in PTP1B, structural data have shown that a secondary binding site can also be found in other PTPs, albeit with varying degrees of accessibility. Along with improvements in pTyr mimetics, we believe that the future will see an increase in the number of orally bioavailable bidentate inhibitors against the various classes of PTPs.