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Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in â¼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.
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Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/enzimología , Neoplasias/genética , Proteínas Tirosina Quinasas Receptoras/genética , Iniciación de la Transcripción Genética , Alelos , Quinasa de Linfoma Anaplásico , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Femenino , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Intrones/genética , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/genética , Lisina/metabolismo , Metilación , Ratones , Datos de Secuencia Molecular , Peso Molecular , Células 3T3 NIH , Neoplasias/tratamiento farmacológico , Oncogenes/genética , Estructura Terciaria de Proteína/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/química , Transducción de SeñalRESUMEN
A subgroup of castration-resistant prostate cancer (CRPC) aberrantly expresses a gastrointestinal (GI) transcriptome governed by two GI-lineage-restricted transcription factors, HNF1A and HNF4G. In this study, we found that expression of GI transcriptome in CRPC correlates with adverse clinical outcomes to androgen receptor signaling inhibitor treatment and shorter overall survival. Bromo- and extra-terminal domain inhibitors (BETi) downregulated HNF1A, HNF4G, and the GI transcriptome in multiple CRPC models, including cell lines, patient-derived organoids, and patient-derived xenografts, while AR and the androgen-dependent transcriptome were largely spared. Accordingly, BETi selectively inhibited growth of GI transcriptome-positive preclinical models of prostate cancer. Mechanistically, BETi inhibited BRD4 binding at enhancers globally, including both AR and HNF4G bound enhancers while gene expression was selectively perturbed. Restoration of HNF4G expression in the presence of BETi rescued target gene expression without rescuing BRD4 binding. This suggests that inhibition of master transcription factors expression underlies the selective transcriptional effects of BETi. SIGNIFICANCE: GI transcriptome expression in CRPC is regulated by the HNF1A-HNF4G-BRD4 axis and correlates with worse clinical outcomes. Accordingly, BET inhibitors significantly reduce tumor cell growth in multiple GI-transcriptome-positive preclinical models of CRPC. Our studies point that expression of GI transcriptome could serve as a predictive biomarker to BETi therapy response.
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We recently demonstrated that CDR1 overexpression in azole-resistant isolates of Candida albicans is due to its enhanced transcriptional activation and increased mRNA stability. In this study, we provide the first evidence of transcriptional regulation of CDR1 by Ncb2, the ß subunit of NC2, a heterodimeric regulator of transcription. Conditional NCB2 null mutants displayed decreased susceptibility toward azole and an enhanced transcription of CDR1. Interestingly, Ncb2 associated with the CDR1 promoter under both repression and activation; however, an increase in recruitment was observed under both transient and constitutive activation states. By chromatin immunoprecipitation (ChIP) assay, we showed the preferential recruitment of Ncb2 to the core TATA region under activation (azole-resistant isolate), while under repression (azole-susceptible isolate) it was present at the TATA upstream region. Further, ChIP analysis revealed that Ncb2 binding was not restricted to the CDR1 gene; instead, it was observed on the promoters of genes coregulated with CDR1 by the transcription activator Tac1. The tac1Δ null mutants, which fail to show the drug-induced transient activation of CDR1, also showed no increase in Ncb2 recruitment at the promoter. Taken together, our results show that Ncb2, in conjunction with Tac1, is involved in the transcriptional activation of CDR1, opening up new therapeutic possibilities to combat multidrug resistance (MDR) in C. albicans.
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Antifúngicos/farmacología , Azoles/farmacología , Candida albicans/metabolismo , Candidiasis/microbiología , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Candida albicans/efectos de los fármacos , Candida albicans/genética , Candida albicans/aislamiento & purificación , Farmacorresistencia Fúngica , Proteínas Fúngicas/metabolismo , Humanos , Proteínas de Transporte de Membrana/metabolismo , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
The comparative analysis of the fatty acid composition of Cassia tora (leaves and stem) was determined using gas chromatography-mass spectrometry. Twenty-seven fatty acids were identified in C. tora (leaves and stem) which was collected from three different geographical areas of India: Lucknow (Uttar Pradesh), Nainital (Uttarakhand), and Bhavnagar (Gujarat), coded as CT-1, CT-2, and CT-3, respectively. The gas chromatography-mass spectrometry analysis showed the presence of various saturated and unsaturated fatty acids. The major fatty acids found were palmitic acid, linoleic acid, linolenic acid, margaric acid, melissic acid, and behenic acid. The highest amounts of saturated fatty acids were found in leaves of C. tora collected from Bhavnagar (Gujarat) (60.7% ± 0.5%). Thus, the study reveals that C. tora has a major amount of nutritionally important fatty acids, along with significant antimicrobial potential. Fatty acids play a significant role in the development of fat products with enhanced nutritional value and clinical application. Remarkable differences were found in the present study between fatty acid profiles of C. tora collected from different locations in India. To the best of our knowledge there is no previously reported comparative study of the fatty acids of C. tora.
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Antibacterianos/química , Antibacterianos/farmacología , Cassia/química , Ácidos Grasos/química , Extractos Vegetales/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Tallos de la Planta/química , Bacterias/efectos de los fármacos , Cromatografía de Gases y Espectrometría de Masas , Geografía , Humanos , India , Pruebas de Sensibilidad MicrobianaRESUMEN
The cellular context that integrates upstream signaling and downstream nuclear response dictates the oncogenic behavior and shapes treatment responses in distinct cancer types. Here, we uncover that in gastrointestinal stromal tumor (GIST), the forkhead family member FOXF1 directly controls the transcription of two master regulators, KIT and ETV1, both required for GIST precursor-interstitial cells of Cajal lineage specification and GIST tumorigenesis. Further, FOXF1 colocalizes with ETV1 at enhancers and functions as a pioneer factor that regulates the ETV1-dependent GIST lineage-specific transcriptome through modulation of the local chromatin context, including chromatin accessibility, enhancer maintenance, and ETV1 binding. Functionally, FOXF1 is required for human GIST cell growth in vitro and murine GIST tumor growth and maintenance in vivo The simultaneous control of the upstream signaling and nuclear response sets up a unique regulatory paradigm and highlights the critical role of FOXF1 in enforcing the GIST cellular context for highly lineage-restricted clinical behavior and treatment response.Significance: We uncover that FOXF1 defines the core-regulatory circuitry in GIST through both direct transcriptional regulation and pioneer factor function. The unique and simultaneous control of signaling and transcriptional circuitry by FOXF1 sets up an enforced transcriptional addiction to FOXF1 in GIST, which can be exploited diagnostically and therapeutically. Cancer Discov; 8(2); 234-51. ©2017 AACR.See related commentary by Lee and Duensing, p. 146This article is highlighted in the In This Issue feature, p. 127.
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Factores de Transcripción Forkhead/genética , Tumores del Estroma Gastrointestinal/genética , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Animales , Biomarcadores de Tumor , Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Tumores del Estroma Gastrointestinal/metabolismo , Perfilación de la Expresión Génica , Xenoinjertos , Humanos , Unión Proteica , Transducción de Señal , Factores de Transcripción/genética , TranscriptomaRESUMEN
Aberrant activation of MAPK signaling leads to the activation of oncogenic transcriptomes. How MAPK signaling is coupled with the transcriptional response in cancer is not fully understood. In 2 MAPK-activated tumor types, gastrointestinal stromal tumor and melanoma, we found that ETV1 and other Pea3-ETS transcription factors are critical nuclear effectors of MAPK signaling that are regulated through protein stability. Expression of stabilized Pea3-ETS factors can partially rescue the MAPK transcriptome and cell viability after MAPK inhibition. To identify the players involved in this process, we performed a pooled genome-wide RNAi screen using a fluorescence-based ETV1 protein stability sensor and identified COP1, DET1, DDB1, UBE3C, PSMD4, and COP9 signalosome members. COP1 or DET1 loss led to decoupling between MAPK signaling and the downstream transcriptional response, where MAPK inhibition failed to destabilize Pea3 factors and fully inhibit the MAPK transcriptome, thus resulting in decreased sensitivity to MAPK pathway inhibitors. We identified multiple COP1 and DET1 mutations in human tumors that were defective in the degradation of Pea3-ETS factors. Two melanoma patients had de novo DET1 mutations arising after vemurafenib treatment. These observations indicate that MAPK signaling-dependent regulation of Pea3-ETS protein stability is a key signaling node in oncogenesis and therapeutic resistance to MAPK pathway inhibition.
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Proteínas Portadoras/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/metabolismo , Mutación , Proteínas Proto-Oncogénicas c-ets/metabolismo , Transcriptoma/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Vemurafenib/farmacología , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/patología , Ratones , Ratones SCID , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma/genética , Ubiquitina-Proteína Ligasas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gastrointestinal stromal tumor (GIST) is the most common subtype of sarcoma. Despite clinical advances in the treatment of KIT/PDGFRA-mutant GIST, similar progress against KIT/PDGFRA wild-type GIST, including mutant BRAF-driven tumors, has been limited by a lack of model systems. ETV1 is a master regulator in the intestinal cells of Cajal (ICC), thought to be the cells of origin of GIST. Here, we present a model in which the ETV1 promoter is used to specifically and inducibly drive Cre recombinase in ICC as a strategy to study GIST pathogenesis. Using a conditional allele for BrafV600E , a mutation observed in clinical cases of GIST, we observed that BrafV600E activation was sufficient to drive ICC hyperplasia but not GIST tumorigenesis. In contrast, combining BrafV600E activation with Trp53 loss was sufficient to drive both ICC hyperplasia and formation of multifocal GIST-like tumors in the mouse gastrointestinal tract with 100% penetrance. This mouse model of sporadic GIST model was amenable to therapeutic intervention, and it recapitulated clinical responses to RAF inhibition seen in human GIST. Our work offers a useful in vivo model of human sporadic forms of BRAF-mutant GIST to help unravel its pathogenesis and therapeutic response to novel experimental agents. Cancer Res; 77(14); 3758-65. ©2017 AACR.
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Proteínas de Unión al ADN/genética , Proteínas Proto-Oncogénicas B-raf/genética , Factores de Transcripción/genética , Animales , Modelos Animales de Enfermedad , Neoplasias Gastrointestinales/enzimología , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/patología , Tumores del Estroma Gastrointestinal/enzimología , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/patología , Ratones , Ratones SCID , MutaciónRESUMEN
Prostate cancer exhibits a lineage-specific dependence on androgen signaling. Castration resistance involves reactivation of androgen signaling or activation of alternative lineage programs to bypass androgen requirement. We describe an aberrant gastrointestinal-lineage transcriptome expressed in â¼5% of primary prostate cancer that is characterized by abbreviated response to androgen-deprivation therapy and in â¼30% of castration-resistant prostate cancer. This program is governed by a transcriptional circuit consisting of HNF4G and HNF1A. Cistrome and chromatin analyses revealed that HNF4G is a pioneer factor that generates and maintains enhancer landscape at gastrointestinal-lineage genes, independent of androgen-receptor signaling. In HNF4G/HNF1A-double-negative prostate cancer, exogenous expression of HNF4G at physiologic levels recapitulates the gastrointestinal transcriptome, chromatin landscape, and leads to relative castration resistance.
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Resistencia a Antineoplásicos/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Animales , Xenoinjertos , Humanos , Masculino , Ratones , Ratones SCID , Neoplasias de la Próstata Resistentes a la Castración/patología , Inhibidor de Tripsina Pancreática de Kazal/biosíntesisRESUMEN
UNLABELLED: Gastrointestinal stromal tumor (GIST), originating from the interstitial cells of Cajal (ICC), is characterized by frequent activating mutations of the KIT receptor tyrosine kinase. Despite the clinical success of imatinib, which targets KIT, most patients with advanced GIST develop resistance and eventually die of the disease. The ETS family transcription factor ETV1 is a master regulator of the ICC lineage. Using mouse models of Kit activation and Etv1 ablation, we demonstrate that ETV1 is required for GIST initiation and proliferation in vivo, validating it as a therapeutic target. We further uncover a positive feedback circuit where MAP kinase activation downstream of KIT stabilizes the ETV1 protein, and ETV1 positively regulates KIT expression. Combined targeting of ETV1 stability by imatinib and MEK162 resulted in increased growth suppression in vitro and complete tumor regression in vivo. The combination strategy to target ETV1 may provide an effective therapeutic strategy in GIST clinical management. SIGNIFICANCE: ETV1 is a lineage-specific oncogenic transcription factor required for the growth and survival of GIST. We describe a novel strategy of targeting ETV1 protein stability by the combination of MEK and KIT inhibitors that synergistically suppress tumor growth. This strategy has the potential to change first-line therapy in GIST clinical management.
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Antineoplásicos/farmacología , Proteínas de Unión al ADN/metabolismo , Tumores del Estroma Gastrointestinal/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Studies of ETS-mediated prostate oncogenesis have been hampered by a lack of suitable experimental systems. Here we describe a new conditional mouse model that shows robust, homogenous ERG expression throughout the prostate. When combined with homozygous Pten loss, the mice developed accelerated, highly penetrant invasive prostate cancer. In mouse prostate tissue, ERG markedly increased androgen receptor (AR) binding. Robust ERG-mediated transcriptional changes, observed only in the setting of Pten loss, included the restoration of AR transcriptional output and upregulation of genes involved in cell death, migration, inflammation and angiogenesis. Similarly, ETS variant 1 (ETV1) positively regulated the AR cistrome and transcriptional output in ETV1-translocated, PTEN-deficient human prostate cancer cells. In two large clinical cohorts, expression of ERG and ETV1 correlated with higher AR transcriptional output in PTEN-deficient prostate cancer specimens. We propose that ETS factors cause prostate-specific transformation by altering the AR cistrome, priming the prostate epithelium to respond to aberrant upstream signals such as PTEN loss.
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Transformación Celular Neoplásica/patología , Genes/genética , Fosfohidrolasa PTEN/deficiencia , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-ets/metabolismo , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Ratones , Proteínas Oncogénicas/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fenotipo , Análisis de Componente Principal , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/genética , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Regulador Transcripcional ERG , Transcriptoma/genéticaRESUMEN
ABSTRACT Roccella montagnei Bél. belongs to lichen family Roccelleceae growing luxuriantly along the coastal regions of India. As Roccella has been shown to be bioactive, we prepared methanolic extract and assessed its anticancer potential. The methanolic extract showed significant in vitro cytotoxic activity against four human cancer cell lines such as colon (DLD-1, SW-620), breast (MCF-7), head and neck (FaDu). This prompted us to isolate bioactive compounds through column chromatography. Two compounds roccellic acid and everninic acid have been isolated, out of which everninic acid is reported for the first time. Both the compounds have been tested for in vitro cytotoxic activity in which roccellic acid showed strong anticancer activity as compared to the everninic acid. Cyclin Dependent Kinase (CDK-10) contributes to proliferation of cancer cells, and aberrant activity of these kinases has been reported in a wide variety of human cancers. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics. Therefore both the isolated compounds were tested for in silico molecular docking study against Cyclin Dependent Kinase isomer enzyme to support the cytotoxic activity.
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Candida drug resistance protein (Cdr1p) is a major drug efflux protein, which plays a key role in commonly encountered clinical azole resistance in Candida albicans. We have analyzed its sequence in several azole resistant clinical isolates to evaluate the allelic variation within CDR1 gene and to relate it to its functional activity. The sequence analysis revealed 53 single nucleotide polymorphisms (SNPs), out of which six were non-synonymous single nucleotide polymorphisms (NS-SNPs) implying a change in amino acid and were found in two or more than two allelic combinations in different sensitive or resistant isolates. We have identified three new NS-SNPs namely, E948P, T950S, and F1399Y, in isolates wherein F1399Y appeared to be unique and was present in one of the naturally occurring azole resistant isolates obtained from Indian diabetic patients. However, site-directed mutagenesis showed that the residue F1399 in between TMS 11 and TMS 12 does not affect the functionality of Cdr1p. Taken together, our SNPs analyses reveal that unlike human P-gp, the naturally acquired allelic variations are mostly present in non-conserved regions of the protein which do not allow Cdr1p to genetically evolve in a manner, that would allow a change in its functionality to affect substrate recognition, specificity, and drug efflux activity of C. albicans cells.