RESUMEN
Cancer cells depend on transcription of telomerase reverse transcriptase (TERT). Many transcription factors affect TERT, though regulation occurs in context of a broader network. Network effects on telomerase regulation have not been investigated, though deeper understanding of TERT transcription requires a systems view. However, control over individual interactions in complex networks is not easily achievable. Mathematical modelling provides an attractive approach for analysis of complex systems and some models may prove useful in systems pharmacology approaches to drug discovery. In this report, we used transfection screening to test interactions among 14 TERT regulatory transcription factors and their respective promoters in ovarian cancer cells. The results were used to generate a network model of TERT transcription and to implement a dynamic Boolean model whose steady states were analysed. Modelled effects of signal transduction inhibitors successfully predicted TERT repression by Src-family inhibitor SU6656 and lack of repression by ERK inhibitor FR180204, results confirmed by RT-QPCR analysis of endogenous TERT expression in treated cells. Modelled effects of GSK3 inhibitor 6-bromoindirubin-3'-oxime (BIO) predicted unstable TERT repression dependent on noise and expression of JUN, corresponding with observations from a previous study. MYC expression is critical in TERT activation in the model, consistent with its well known function in endogenous TERT regulation. Loss of MYC caused complete TERT suppression in our model, substantially rescued only by co-suppression of AR. Interestingly expression was easily rescued under modelled Ets-factor gain of function, as occurs in TERT promoter mutation. RNAi targeting AR, JUN, MXD1, SP3, or TP53, showed that AR suppression does rescue endogenous TERT expression following MYC knockdown in these cells and SP3 or TP53 siRNA also cause partial recovery. The model therefore successfully predicted several aspects of TERT regulation including previously unknown mechanisms. An extrapolation suggests that a dominant stimulatory system may programme TERT for transcriptional stability.
Asunto(s)
Modelos Genéticos , Neoplasias/enzimología , Neoplasias/genética , Telomerasa/antagonistas & inhibidores , Telomerasa/genética , Línea Celular Tumoral , Biología Computacional , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Femenino , Redes Reguladoras de Genes/genética , Genes myc , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Humanos , Indoles/farmacología , Conceptos Matemáticos , Modelos Biológicos , Neoplasias/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/genética , Proteína Proto-Oncogénica c-ets-2/genética , Pirazoles/farmacología , Piridazinas/farmacología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Telomerasa/metabolismo , TransfecciónRESUMEN
Protein kinase C iota (PKCι), a serine/threonine kinase required for cell polarity, proliferation and migration, is commonly up- or downregulated in cancer. PKCι is a human oncogene but whether this is related to its role in cell polarity and what repertoire of oncogenes acts in concert with PKCι is not known. We developed a panel of candidate oncogene expressing Madin-Darby canine kidney (MDCK) cells and demonstrated that H-Ras, ErbB2 and phosphatidylinositol 3-kinase transformation led to non-polar spheroid morphogenesis (dysplasia), whereas MDCK spheroids expressing c-Raf or v-Src were largely polarized. We show that small interfering RNA (siRNA)-targeting PKCι decreased the size of all spheroids tested and partially reversed the aberrant polarity phenotype in H-Ras and ErbB2 spheroids only. This indicates distinct requirements for PKCι and moreover that different thresholds of PKCι activity are required for these phenotypes. By manipulating PKCι function using mutant constructs, siRNA depletion or chemical inhibition, we have demonstrated that PKCι is required for polarization of parental MDCK epithelial cysts in a 3D matrix and that there is a threshold of PKCι activity above and below which, disorganized epithelial morphogenesis results. Furthermore, treatment with a novel PKCι inhibitor, CRT0066854, was able to restore polarized morphogenesis in the dysplastic H-Ras spheroids. These results show that tightly regulated PKCι is required for normal-polarized morphogenesis in mammalian cells and that H-Ras and ErbB2 cooperate with PKCι for loss of polarization and dysplasia. The identification of a PKCι inhibitor that can restore polarized morphogenesis has implications for the treatment of Ras and ErbB2 driven malignancies.
Asunto(s)
Polaridad Celular , Transformación Celular Neoplásica/patología , Quistes/patología , Células Epiteliales/patología , Isoenzimas/metabolismo , Morfogénesis/fisiología , Proteína Quinasa C/metabolismo , Esferoides Celulares/patología , Animales , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Quistes/metabolismo , Perros , Células Epiteliales/metabolismo , Genes ras/fisiología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Riñón/metabolismo , Riñón/patología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , ARN Interferente Pequeño/genética , Receptor ErbB-2/metabolismo , Esferoides Celulares/metabolismoRESUMEN
The aPKC [atypical PKC (protein kinase C)] isoforms ι and ζ play crucial roles in the formation and maintenance of cell polarity and represent attractive anti-oncogenic drug targets in Ras-dependent tumours. To date, few isoform-specific chemical biology tools are available to inhibit aPKC catalytic activity. In the present paper, we describe the identification and functional characterization of potent and selective thieno[2,3-d]pyrimidine-based chemical inhibitors of aPKCs. A crystal structure of human PKCι kinase domain bound to a representative compound, CRT0066854, reveals the basis for potent and selective chemical inhibition. Furthermore, CRT0066854 displaces a crucial Asn-Phe-Asp motif that is part of the adenosine-binding pocket and engages an acidic patch used by arginine-rich PKC substrates. We show that CRT0066854 inhibits the LLGL2 (lethal giant larvae 2) phosphorylation in cell lines and exhibits phenotypic effects in a range of cell-based assays. We conclude that this compound can be used as a chemical tool to modulate aPKC activity in vitro and in vivo and may guide the search for further aPKC-selective inhibitors.
Asunto(s)
Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/química , Tiofenos/farmacología , Adenosina/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Proteínas del Citoesqueleto/metabolismo , Perros , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Isoenzimas/antagonistas & inhibidores , Imitación Molecular , Datos de Secuencia Molecular , Fosforilación , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Pirimidinas/farmacología , Tiofenos/químicaRESUMEN
BACKGROUND: Cellular senescence is a major barrier to tumour progression, though its role in pathogenesis of cancer and other diseases is poorly understood in vivo. Improved understanding of the degree to which latent senescence signalling persists in tumours might identify intervention strategies to provoke "accelerated senescence" responses as a therapeutic outcome. Senescence involves convergence of multiple pathways and requires ongoing dynamic signalling throughout its establishment and maintenance. Recent discovery of several new markers allows for an expression profiling approach to study specific senescence phenotypes in relevant tissue samples. We adopted a "senescence scoring" methodology based on expression profiles of multiple senescence markers to examine the degree to which signals of damage-associated or secretory senescence persist in various human tumours. RESULTS: We first show that scoring captures differential induction of damage or inflammatory pathways in a series of public datasets involving radiotherapy of colon adenocarcinoma, chemotherapy of breast cancer cells, replicative senescence of mesenchymal stem cells, and progression of melanoma. We extended these results to investigate correlations between senescence score and growth inhibition in response to ~1500 compounds in the NCI60 panel. Scoring of our own mesenchymal tumour dataset highlighted differential expression of secretory signalling pathways between distinct subgroups of MPNST, liposarcomas and peritoneal mesothelioma. Furthermore, a pro-inflammatory signature yielded by hierarchical clustering of secretory markers showed prognostic significance in mesothelioma. CONCLUSIONS: We find that "senescence scoring" accurately reports senescence signalling in a variety of situations where senescence would be expected to occur and highlights differential expression of damage associated and secretory senescence pathways in a context-dependent manner.
Asunto(s)
Antineoplásicos/toxicidad , Senescencia Celular/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inflamación/genética , Mesotelioma/genética , Neoplasias Peritoneales/genética , Transducción de Señal/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Análisis por Conglomerados , Bases de Datos Genéticas , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Inflamación/complicaciones , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Mesotelioma/tratamiento farmacológico , Mesotelioma/patología , Mesotelioma/radioterapia , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Peritoneales/patología , Neoplasias Peritoneales/radioterapia , Pronóstico , Proyectos de Investigación , Transducción de Señal/efectos de los fármacos , Análisis de SupervivenciaRESUMEN
Telomere signaling and metabolic dysfunction are hallmarks of cell aging. New agents targeting these processes might provide therapeutic opportunities, including chemoprevention strategies against cancer predisposition. We report identification and characterization of a pyrazolopyrimidine compound series identified from screens focused on cell immortality and whose targets are glycolytic kinase PGK1 and oxidative stress sensor DJ1. We performed structure-activity studies on the series to develop a photoaffinity probe to deconvolute the cellular targets. In vitro binding and structural analyses confirmed these targets, suggesting that PGK1/DJ1 interact, which we confirmed by immunoprecipitation. Glucose homeostasis and oxidative stress are linked to telomere signaling and exemplar compound CRT0063465 blocked hypoglycemic telomere shortening. Intriguingly, PGK1 and DJ1 bind to TRF2 and telomeric DNA. Compound treatment modulates these interactions and also affects Shelterin complex composition, while conferring cellular protection from cytotoxicity due to bleomycin and desferroxamine. These results demonstrate therapeutic potential of the compound series.
Asunto(s)
Complejos Multiproteicos/metabolismo , Fosfoglicerato Quinasa/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Estrés Fisiológico , Homeostasis del Telómero/efectos de los fármacos , Proteínas de Unión a Telómeros/metabolismo , Línea Celular Tumoral , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Complejos Multiproteicos/química , Fosfoglicerato Quinasa/química , Unión Proteica , Proteína Desglicasa DJ-1/química , Pirazoles/síntesis química , Pirazoles/química , Pirimidinas/síntesis química , Pirimidinas/química , Complejo Shelterina , Relación Estructura-Actividad , Telómero/genética , Telómero/metabolismo , Acortamiento del Telómero/efectos de los fármacos , Acortamiento del Telómero/genética , Proteínas de Unión a Telómeros/químicaRESUMEN
The conserved polarity effector proteins PAR-3, PAR-6, CDC-42, and atypical protein kinase C (aPKC) form a core unit of the PAR protein network, which plays a central role in polarizing a broad range of animal cell types. To functionally polarize cells, these proteins must activate aPKC within a spatially defined membrane domain on one side of the cell in response to symmetry-breaking cues. Using the Caenorhabditis elegans zygote as a model, we find that the localization and activation of aPKC involve distinct, specialized aPKC-containing assemblies: a PAR-3-dependent assembly that responds to polarity cues and promotes efficient segregation of aPKC toward the anterior but holds aPKC in an inactive state, and a CDC-42-dependent assembly in which aPKC is active but poorly segregated. Cycling of aPKC between these distinct functional assemblies, which appears to depend on aPKC activity, effectively links cue-sensing and effector roles within the PAR network to ensure robust establishment of polarity.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Polaridad Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Cigoto/metabolismoRESUMEN
Advanced basal cell carcinomas (BCCs) circumvent Smoothened (SMO) inhibition by activating GLI transcription factors to sustain the high levels of Hedgehog (HH) signaling required for their survival. Unfortunately, there is a lack of efficacious therapies. We performed a gene expression-based drug repositioning screen in silico and identified the FDA-approved histone deacetylase (HDAC) inhibitor, vorinostat, as a top therapeutic candidate. We show that vorinostat only inhibits proliferation of BCC cells in vitro and BCC allografts in vivo at high dose, limiting its usefulness as a monotherapy. We leveraged this in silico approach to identify drug combinations that increase the therapeutic window of vorinostat and identified atypical PKC Æ/Ê (aPKC) as a HDAC costimulator of HH signaling. We found that aPKC promotes GLI1-HDAC1 association in vitro, linking two positive feedback loops. Combination targeting of HDAC1 and aPKC robustly inhibited GLI1, lowering drug doses needed in vitro, in vivo, and ex vivo in patient-derived BCC explants. We identified a bioavailable and selective small-molecule aPKC inhibitor, bringing the pharmacological blockade of aPKC and HDAC1 into the realm of clinical possibility. Our findings provide a compelling rationale and candidate drugs for combined targeting of HDAC1 and aPKC in HH-dependent cancers.
Asunto(s)
Carcinoma Basocelular/tratamiento farmacológico , Histona Desacetilasa 1/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Isoenzimas/efectos de los fármacos , Proteína Quinasa C/efectos de los fármacos , Neoplasias Cutáneas/tratamiento farmacológico , Aloinjertos , Animales , Carcinoma Basocelular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Biología Computacional , Combinación de Medicamentos , Descubrimiento de Drogas , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Erizos/genética , Erizos/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Inhibidores de Histona Desacetilasas/química , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa C/metabolismo , Transducción de Señal , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Translating existing and emerging knowledge of cancer biology into effective novel therapies remains a great challenge in drug discovery. A firm understanding of the target biology, confidence in the supporting preclinical research, and access to diverse chemical matter is required to lower attrition rates and prosecute targets effectively. Understanding past successes and failures will aid in refining this process to deliver further therapeutic benefit to patients. In this review, we suggest that early oncology drug discovery should focus on selection and prosecution of cancer targets with strong disease biology rather than on more chemically "druggable" targets with only modest disease-linkage. This approach offers higher potential benefit but also increases the need for innovative and alternative approaches. These include using different methods to validate novel targets and identify chemical matter, as well as raising the standards and our interpretation of the scientific literature. The combination of skills required for this emphasizes the need for broader early collaborations between academia and industry.
Asunto(s)
Descubrimiento de Drogas , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Academias e Institutos , Animales , Conducta Cooperativa , Descubrimiento de Drogas/métodos , Industria Farmacéutica , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Senescence forms a universal block to tumorigenesis which impacts on all hallmarks of cancer, making it an attractive target for drug discovery. Therefore a strategy must be devised to focus this broad potential into a manageable drug discovery programme. Several issues remain to be addressed including the lack of robust senescence-inducing compounds and causally related biomarkers to measure cellular response. Here, we review the latest progress in translating senescence as a target for cancer therapy and some promising approaches to drug and biomarker discovery. Finally, we discuss the potential application of a senescence-induction therapy in a clinical setting.
Asunto(s)
Antineoplásicos/farmacología , Senescencia Celular/efectos de los fármacos , Senescencia Celular/fisiología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Animales , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Senescencia Celular/genética , Descubrimiento de Drogas/métodos , Humanos , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
Intervention in protein kinase C (PKC) has a chequered history, partly because of the poor selectivity of many inhibitors and partly a reflection of the sometimes antagonistic action of related PKC isoforms. Recent advances in targeting PKC isoforms have come from structural work on isolated kinase domains that have provided opportunities to drive selectivity through structure-based avenues. The promise of isoform selective inhibitors and the rationale for their development are discussed in the broader context of the PKC inhibitor arsenal.
Asunto(s)
Isoenzimas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Animales , Dominio Catalítico , Ensayos Clínicos como Asunto , Activación Enzimática , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Estructura Molecular , Familia de Multigenes , Neoplasias/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Telomerase controls telomere homeostasis and cell immortality and is a promising anti-cancer target, but few small molecule telomerase inhibitors have been developed. Reactivated transcription of the catalytic subunit hTERT in cancer cells controls telomerase expression. Better understanding of upstream pathways is critical for effective anti-telomerase therapeutics and may reveal new targets to inhibit hTERT expression. METHODOLOGY/PRINCIPAL FINDINGS: In a focused promoter screen, several GSK3 inhibitors suppressed hTERT reporter activity. GSK3 inhibition using 6-bromoindirubin-3'-oxime suppressed hTERT expression, telomerase activity and telomere length in several cancer cell lines and growth and hTERT expression in ovarian cancer xenografts. Microarray analysis, network modelling and oligonucleotide binding assays suggested that multiple transcription factors were affected. Extensive remodelling involving Sp1, STAT3, c-Myc, NFkappaB, and p53 occurred at the endogenous hTERT promoter. RNAi screening of the hTERT promoter revealed multiple kinase genes which affect the hTERT promoter, potentially acting through these factors. Prolonged inhibitor treatments caused dynamic expression both of hTERT and of c-Jun, p53, STAT3, AR and c-Myc. CONCLUSIONS/SIGNIFICANCE: Our results indicate that GSK3 activates hTERT expression in cancer cells and contributes to telomere length homeostasis. GSK3 inhibition is a clinical strategy for several chronic diseases. These results imply that it may also be useful in cancer therapy. However, the complex network effects we show here have implications for either setting.