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Our previous studies determined that elevating SOX2 in a wide range of tumor cells leads to a reversible state of tumor growth arrest. Efforts to understand how tumor cell growth is inhibited led to the discovery of a SOX2:MYC axis that is responsible for downregulating c-MYC (MYC) when SOX2 is elevated. Although we had determined that elevating SOX2 downregulates MYC transcription, the mechanism responsible was not determined. Given the challenges of targeting MYC clinically, we set out to identify how elevating SOX2 downregulates MYC transcription. In this study, we focused on the MYC promoter region and an upstream region of the MYC locus that contains a MYC super-enhancer encompassing five MYC enhancers and which is associated with several cancers. Here we report that BRD4 and p300 associate with each of the MYC enhancers in the upstream MYC super-enhancer as well as the MYC promoter region and that elevating SOX2 decreases the recruitment of BRD4 and p300 to these sites. Additionally, we determined that elevating SOX2 leads to increases in the association of SOX2 and H3K27me3 within the MYC super-enhancer and the promoter region of MYC. Importantly, we conclude that the increases in SOX2 within the MYC super-enhancer precipitate a cascade of events that culminates in the repression of MYC transcription. Together, our studies identify a novel molecular mechanism able to regulate MYC transcription in two distinctly different tumor types and provide new mechanistic insights into the molecular interrelationships between two master regulators, SOX2 and MYC, widely involved in multiple cancers.
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SOX10 is a lineage-specific transcription factor critical for melanoma tumor growth, while SOX10 loss-of-function drives the emergence of therapy-resistant, invasive melanoma phenotypes. A major challenge has been developing therapeutic strategies targeting SOX10's role in melanoma proliferation, while preventing a concomitant increase in tumor cell invasion. Here, we report that the lysine acetyltransferase (KAT) EP300 and SOX10 gene loci on Chromosome 22 are frequently co-amplified in melanomas, including UV-associated and acral tumors. We further show that p300 KAT activity mediates SOX10 protein stability and that the p300 inhibitor, A-485, downregulates SOX10 protein levels in melanoma cells via proteasome-mediated degradation. Additionally, A-485 potently inhibits proliferation of SOX10+ melanoma cells while decreasing invasion in AXLhigh/MITFlow melanoma cells through downregulation of metastasis-related genes. We conclude that the SOX10/p300 axis is critical to melanoma growth and invasion, and that inhibition of p300 KAT activity through A-485 may be a worthwhile therapeutic approach for SOX10-reliant tumors.
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The small molecule A-485 competitively inhibits the histone acetyltransferase domain of CBP (cyclic-adenosine monophosphate response element-binding protein)/p300. Apart from its antineoplastic activity, researchers are exploring its potential benefits in treating osteoporosis and its impact on energy metabolism. However, so far, only limited pharmacokinetic data are available, and the crucial determination of A-485 concentration in various biological materials with small sample volumes remains unpublished. A rapid and sensitive LC-tandem mass spectrometry method has been developed and validated to quantify A-485 in mouse serum and tissue. In this method, serum samples underwent precipitation with acetonitrile, while cell lysates were appropriately diluted. The determination of A-485 utilized a reversed-phase column with a mobile phase gradient, and detection was carried out in multiple reaction monitoring mode. The lower standard sample, with a concentration of 7.8 ng/mL, served as the lower limit of quantification, while the upper standard was established at 1000 ng/mL. A-485 concentrations were assessed in both serum samples and the lysate of all examined tissues, revealing swift metabolic clearance. The analytical method outlined here is deemed appropriate for subsequent studies. The ability to measure the active ingredient in various compartments facilitates the determination of accurate pharmacokinetic parameters. In the event of human use of A-485, the analysis method can be seamlessly transferred to human samples.
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Antineoplásicos , Espectrometría de Masas en Tándem , Ratones , Animales , Humanos , Espectrometría de Masas en Tándem/métodos , Acetilcoenzima A , Límite de Detección , Cromatografía Liquida/métodosRESUMEN
Elevated hepatic gluconeogenesis is a major contributor of fasting hyperglycemia in diabetes. MicroRNAs (miRNAs) are tightly linked to glucose metabolism, but their role in hepatic gluconeogenesis remains largely unkown. In this current study, miR-34a-5p expression was significantly increased in liver tissues of db/db mice. Overexpression of miR-34a-5p promoted hepatic glucose production in mouse primary hepatocytes with increased expressions of gluconeogenic genes while miR-34a-5p inhibition displayed a contrary action. MiR-34a-5p overexpression in mouse primary hepatocytes repressed SIRT1 expression. SIRT1 inhibition by EX527 blocked phosphoenolpyruvate carboxykinase (PEPCK) protein degradation and enhanced hepatic gluconeogenesis. Treatment of A485 (a CBP/p300 inhibitor) decreased miR-34a-5p and PEPCK expressions in the livers of db/db mice, but elevated SIRT1 protein expression. In mouse primary hepatocytes, A485 exhibited a similar result. Overexpression of miR-34a-5p attenuated A485-inhibited gluconeogenic gene expressions and A485-induced SIRT1 protein expression. Finally, after miR-34a-5p was inhibited in the livers of db/db mice, hepatic glucose production and gluconeogenic gene expressions were markedly lowered. Our findings highlight a critical role of miR-34a-5p in the regulation of hepatic gluconeogenesis and miR-34a-5p may be a potential target in the treatment of type 2 diabetes.
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Diabetes Mellitus Tipo 2 , MicroARNs/genética , Animales , Diabetes Mellitus Tipo 2/genética , Gluconeogénesis/genética , Glucosa/metabolismo , Glucosa/farmacología , Hígado/metabolismo , Ratones , MicroARNs/metabolismo , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
Transient receptor potential M3 (TRPM3) cation channels regulate numerous biological functions, including gene transcription. Stimulation of TRPM3 channels with pregnenolone sulfate activates stimulus-responsive transcription factors, which bind to short cognate sequences in the promoters of their target genes. In addition, coregulator proteins are involved that convert the chromatin into a configuration that is permissive for gene transcription. In this study, we determined whether TRPM3-induced gene transcription requires coactivators that change the acetylation pattern of histones. We used compound A485, a specific inhibitor of the histone acetyltransferases CBP and p300. In addition, the role of bromodomain proteins that bind to acetylated lysine residues of histones was analyzed. We used JQ1, an inhibitor of bromodomain and extra terminal domain (BET) family proteins. The results show that both compounds attenuated the activation of AP-1 and CREB-regulated gene transcription following stimulation of TRPM3 channels. Inhibition of CBP/p300 and BET proteins additionally reduced the transcriptional activation potential of the transcription factors c-Fos and Elk-1. Transcriptional upregulation of the interleukin-8 gene was attenuated by A485 and JQ1, indicating that proinflammatory cytokine expression is controlled by CBP/p300 and bromodomain proteins. We conclude that TRPM3-induced signaling involves transcriptional coactivators and acetyl-lysine-bound bromodomain proteins for activating gene transcription.
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CONTEXT: Growth hormone pituitary adenoma (GHPA), a major subtype of pituitary adenoma (PA), can lead to progressive somatic disfigurement, multiple complications, and even increased mortality. The efficacy of current treatments is limited; thus, a novel pharmacological treatment is urgently needed. As a histone acetyltransferase (HAT) coactivator, p300 can regulate the transcription of several genes that are crucial for PA tumorigenesis and progression. However, the role of p300 and its catalytic inhibitor in GHPA is still unclear. OBJECTIVE: We aimed to identify the expression of p300 in GHPA and in normal pituitary glands. METHODS: The expression of p300 was detected in GHPA and normal pituitary tissues. Genetic knockdown was performed by siRNA. The efficacy of the p300 inhibitor A-485 in the cell cycle, proliferation, apoptosis, and hormone secretion was investigated by flow cytometry, ELISAs, Western blotting, and qRT-PCR. RNA sequencing, bioinformatic analysis, and subsequent validation experiments were performed to reveal the potential biological mechanism of A-485. RESULTS: High expression of p300 was found in GHPA tissues compared with normal pituitary tissues. Knockdown of p300 inhibited cell proliferation and clone formation. Treatment with A-485 suppressed cell growth and inhibited the secretion of GH in vitro and in vivo. Further mechanistic studies showed that A-485 could downregulate the expression or activity of several oncogenes, such as genes in the Pttg1, c-Myc, cAMP and PI3K/AKT/mTOR signaling pathways, which are crucial for PA tumorigenesis and progression. CONCLUSION: Our findings demonstrate that inhibition of HAT p300 by its selective inhibitor A-485 is a promising therapy for GHPA.
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Adenoma , Adenoma Hipofisario Secretor de Hormona del Crecimiento , Hormona de Crecimiento Humana , Neoplasias Hipofisarias , Adenoma/tratamiento farmacológico , Adenoma/genética , Adenoma/metabolismo , Carcinogénesis , Línea Celular Tumoral , Proliferación Celular , Hormona del Crecimiento/uso terapéutico , Adenoma Hipofisario Secretor de Hormona del Crecimiento/genética , Hormona de Crecimiento Humana/uso terapéutico , Humanos , Fosfatidilinositol 3-Quinasas , Neoplasias Hipofisarias/tratamiento farmacológico , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/metabolismoRESUMEN
Estrogen receptor alpha (ER) is the oncogenic driver for ER+ breast cancer (BC). ER antagonists are the standard-of-care treatment for ER+ BC; however, primary and acquired resistance to these agents is common. CBP and p300 are critical ER co-activators and their acetyltransferase (KAT) domain and acetyl-lysine binding bromodomain (BD) represent tractable drug targets, but whether CBP/p300 inhibitors can effectively suppress ER signaling remains unclear. We report that the CBP/p300 KAT inhibitor A-485 and the BD inhibitor GNE-049 downregulate ER, attenuate estrogen-induced c-Myc and Cyclin D1 expression, and inhibit growth of ER+ BC cells through inducing senescence. Microarray and RNA-seq analysis demonstrates that A-485 or EP300 (encoding p300) knockdown globally inhibits expression of estrogen-regulated genes, confirming that ER inhibition is an on-target effect of A-485. Using ChIP-seq, we report that A-485 suppresses H3K27 acetylation in the enhancers of ER target genes (including MYC and CCND1) and this correlates with their decreased expression, providing a mechanism underlying how CBP/p300 inhibition downregulates ER gene network. Together, our results provide a preclinical proof-of-concept that CBP/p300 represent promising therapeutic targets in ER+ BC for inhibiting ER signaling.
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Epigenetics mainly refers to covalent modifications to DNA or histones without affecting genomes, which ultimately lead to phenotypic changes in cells or organisms. Given the abundance of regulatory targets in epigenetic pathways and their pivotal roles in tumorigenesis and drug resistance, the development of epigenetic drugs holds a great promise for the current cancer therapy. However, lack of potent, selective, and clinically tractable small-molecule compounds makes the strategy to target cancer epigenetic pathways still challenging. Therefore, this review focuses on epigenetic pathways, small molecule inhibitors targeting DNA methyltransferase (DNMT) and small molecule inhibitors targeting histone modification (the main regulatory targets are histone acetyltransferases (HAT), histone deacetylases (HDACs) and histone methyltransferases (HMTS)), as well as the combination strategies of the existing epigenetic therapeutic drugs and more new therapies to improve the efficacy, which will shed light on a new clue on discovery of more small-molecule drugs targeting cancer epigenetic pathways as promising strategies in the future.
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Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Animales , Metilasas de Modificación del ADN/antagonistas & inhibidores , Epigénesis Genética , Histonas/metabolismo , Humanos , Resultado del TratamientoRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer caused by the deregulation of key T-cell developmental pathways, including Notch signaling. Aberrant Notch signaling in T-ALL occurs by NOTCH1 gain-of-function mutations and by NOTCH3 overexpression. Although NOTCH3 is assumed as a Notch1 target, machinery driving its transcription in T-ALL is undefined in leukemia subsets lacking Notch1 activation. Here, we found that the binding of the intracellular Notch3 domain, as well as of the activated Notch1 fragment, to the NOTCH3 gene locus led to the recruitment of the H3K27 modifiers JMJD3 and p300, and it was required to preserve transcriptional permissive/active H3K27 marks and to sustain NOTCH3 gene expression levels. Consistently, pharmacological inhibition of JMJD3 by GSKJ4 treatment or of p300 by A-485 decreased the levels of expression of NOTCH3, NOTCH1 and of the Notch target genes DELTEX1 and c-Myc and abrogated cell viability in both Notch1- and Notch3-dependent T-cell contexts. Notably, re-introduction of exogenous Notch1, Notch3 as well as c-Myc partially rescued cells from anti-growth effects induced by either treatment. Overall our findings indicate JMJD3 and p300 as general Notch1 and Notch3 signaling co-activators in T-ALL and suggest further investigation on the potential therapeutic anti-leukemic efficacy of their enzymatic inhibition in Notch/c-Myc axis-related cancers and diseases.
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The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here, we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites and a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (<30 min) acetylation turnover, revealing a dynamic balance between acetylation and deacetylation. Quantification of acetylation, mRNA, and protein abundance after CBP/p300 inhibition reveals a kinetically competent network of gene expression that strictly depends on CBP/p300-catalyzed rapid acetylation. Collectively, our in-depth acetylome analyses reveal systems attributes of CBP/p300 targets, and the resource dataset provides a framework for investigating CBP/p300 functions and for understanding the impact of small-molecule inhibitors targeting its catalytic and bromodomain activities.