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GSEA enrichments for genes changing expression after JQ or CBP30 treatments. (a) Top reactome pathways significantly enriched in upregulated or downregulated genes. Black boxes indicate significant enrichment with P-value

Oligonucleotides used in this study

Inhibitors that prevent the binding of bromodomains to acetylated histones hold therapeutic potential. However, the effects of targeting most of the 60 different bromodomains found in the human proteome remain unexplored. Here, we investigate the molecular mechanisms responsible for the antiproliferative properties of CREBBP/EP300 bromodomain inhibition in ER-negative breast cancer cell lines. We show using genetic and chemical approaches that CREBBP/EP300 bromodomains are critical to support the proliferation of the triple-negative breast cancer cell line MDA-MB-453. Analysis of the transcriptional pathways affected by CREBBP/EP300 bromodomain inhibitors reveals that the expression of genes associated with super-enhancers is downregulated, which in turn are occupied by very high levels of androgen receptor (AR) in MDA-MB-453 cells. Treatment of MDA-MB-453 with CREBBP/EP300 bromodomain inhibitors downregulates the expression of an AR-dependent signature distinctive of breast cancer tumors that express AR and causes a decrease in H3K27ac levels at AR-binding sites. In accordance, in prostate cancer cell lines that express AR CREBBP/EP300 bromodomain inhibitors downregulate the expression of genes bound by AR and associated with super-enhancers. In summary, we report that triple-negative breast cancer cell lines that express AR are particularly sensitive to CREBBP/EP300 bromodomain inhibitors and consequently these inhibitors hold potential to treat this type of cancer.Implications:AR-dependent cancer cell lines are sensitive to CREBBP/EP300 bromodomain inhibitors

Biogenesis of the U5 small nuclear ribonucleoprotein (snRNP) is an essential and highly regulated process. In particular, PRPF8, one of U5 snRNP main components, requires HSP90 working in concert with R2TP, a cochaperone complex containing RUVBL1 and RUVBL2 AAA-ATPases, and additional factors that are still poorly characterized. Here, we use biochemistry, interaction mapping, mass spectrometry and cryoEM to study the role of ZNHIT2 in the regulation of the R2TP chaperone during the biogenesis of PRPF8. ZNHIT2 forms a complex with R2TP which depends exclusively on the direct interaction of ZNHIT2 with the RUVBL1-RUVBL2 ATPases. The cryoEM analysis of this complex reveals that ZNHIT2 alters the conformation and nucleotide state of RUVBL1-RUVBL2, affecting its ATPase activity. We characterized the interactions between R2TP, PRPF8, ZNHIT2, ECD and AAR2 proteins. Interestingly, PRPF8 makes a direct interaction with R2TP and this complex can incorporate ZNHIT2 and other proteins involved in the biogenesis of PRPF8 such as ECD and AAR2. Together, these results show that ZNHIT2 participates in the assembly of the U5 snRNP as part of a network of contacts between assembly factors required for PRPF8 biogenesis and the R2TP-HSP90 chaperone, while concomitantly regulating the structure and nucleotide state of R2TP. Agencia Estatal de Investigación (AEI/10.13039/501100011033), Ministerio de Ciencia e Innovación and co-funded by the European Regional Development Fund (ERDF-UE) [SAF2017-82632-P and PID2020-114429RB-I00 to O.L.]; Autonomous Region of Madrid and co-funded by the European Social Fund and the European Regional Development Fund [Y2018/BIO4747 and P2018/NMT4443 to O.L., and which support the contracts of S.C. and A.G-C.]; Funding for open access charge: Agencia Estatal de Investigación (AEI/10.13039/501100011033), Ministerio de Ciencia e Innovación, co-funded by the European Regional Development Fund (ERDF-UE) [SAF2017-82632-P to O.L.]; S.C. contract is funded by the CNIO Friends Program philanthropic initiative since June 2021. Sí

Inhibitors that prevent the binding of bromodomains to acetylated histones hold therapeutic potential. However, the effects of targeting most of the 60 different bromodomains found in the human proteome remain unexplored. Here, we investigate the molecular mechanisms responsible for the antiproliferative properties of CREBBP/EP300 bromodomain inhibition in ER-negative breast cancer cell lines. We show using genetic and chemical approaches that CREBBP/EP300 bromodomains are critical to support the proliferation of the triple-negative breast cancer cell line MDA-MB-453. Analysis of the transcriptional pathways affected by CREBBP/EP300 bromodomain inhibitors reveals that the expression of genes associated with super-enhancers is downregulated, which in turn are occupied by very high levels of androgen receptor (AR) in MDA-MB-453 cells. Treatment of MDA-MB-453 with CREBBP/EP300 bromodomain inhibitors downregulates the expression of an AR-dependent signature distinctive of breast cancer tumors that express AR and causes a decrease in H3K27ac levels at AR-binding sites. In accordance, in prostate cancer cell lines that express AR CREBBP/EP300 bromodomain inhibitors downregulate the expression of genes bound by AR and associated with super-enhancers. In summary, we report that triple-negative breast cancer cell lines that express AR are particularly sensitive to CREBBP/EP300 bromodomain inhibitors and consequently these inhibitors hold potential to treat this type of cancer. Implications: AR-dependent cancer cell lines are sensitive to CREBBP/EP300 bromodomain inhibitors

DNA hypermethylation has emerged as a molecular biomarker for the evaluation of cancer diagnosis and prognosis. We define a methylation signature of bladder cancer and evaluate whether this profile assesses prognosis of patients. Genome‐wide methylation analysis was performed on 70 tumor and 10 normal bladder samples. Hypermethylation status of 1505 CpGs present in the promoter region of 807 genes was studied. Thirty‐three genes were significantly hypermethylated in ≥10% of the tumors. Three clusters of patients were characterized by their DNA methylation profile, one at higher risk of dead of disease (p = 0.0012). Association between cluster distribution and stage (p = 0.02) or grade (p = 0.02) was demonstrated. Hypermethylation of JAK3 and absence of hypermethylation of EYA4, GAT6, and SOX1 were associated with low‐grade non‐invasive disease. On the other hand, in high‐grade invasive disease hypermethylation of CSPG2, HOXA11, HOXA9, HS3ST2, SOX1, and TWIST1 was associated with muscle invasiveness. A panel of hypermethylated genes including APC, CSPG2, EPHA5, EYA4, HOXA9, IPF1, ISL1, JAK3, PITX2, SOX1, and TWIST1 predicted cancer‐specific survival and SOX1 (HR = 3.46), PITX2 (HR = 4.17), CSPG2 (HR = 5.35), and JAK3 hypermethylation (HR = 0.19) did so independently. Silencing of genes by hypermethylation is a common event in bladder cancer and could be used to develop diagnostic and prognostic markers. Combined hypermethylation of SOX1, PITX2, or CSPG2 signals patients at higher risk of death from bladder cancer. Sin financiación 2.026 JCR (2017) Q2, 38/79 Pathology; Q3, 83/125 Microbiology; Q4, 124/155 Immunology UEM

Objective To analyze, in older patients with schizophrenia, the methylation status of a set of genes associated with the pathophysiology of the disorder but including anatomical, clinical, and cognitive criteria in the experimental design that, in conjunction with the epigenetic status of specific genes, allows us to derive an integrative model. Method This study included 29 human brain samples from older schizophrenic patients with severe and mild cognitive impairment. We administered a comprehensive battery of neurocognitive tests to determine the size of the impairment across different cognitive domains. We focused our study on the analysis of the methylation pattern of 19 genes of major neurotransmitter systems using methylation-specific PCR and bisulfite genomic sequencing. Results Our results highlight an absence of hypermethylation and hypomethylation in older patients with schizophrenia and in healthy controls, irrespective of the degree of the cognitive deficit measured in the neuropsychological assessment (Fisher's exact test; p

BACKGROUND DNA hypermethylation has emerged as a novel molecular biomarker for the evaluation of prostate cancer diagnosis and prognosis. Defining the specific gene hypermethylation profile for prostate cancer could involve groups of genes that specifically discriminate patients with indolent and aggressive tumors. METHODS Genome-wide methylation analysis was performed on 83 tumor and 10 normal prostate samples using the GoldenGate Methylation Cancer Panel I (Illumina, Inc.). All clinical stages of disease were considered. RESULTS We found 41 genes hypermethylated in more than 20% of the tumors analyzed (P

AKT isoforms are expressed in prostate cancer and their expression and localization have different associations with clinical characteristics. However, the distinct roles of the AKT isoforms in prostate cancer cells are largely unknown. In the present study, we demonstrate distinct roles for AKT1 and AKT2 in cell growth and migration. Ablation of AKT1 and AKT2 decreased the proliferation of the androgen-independent cell line PC-3, although by different mechanisms. AKT1 ablation induced loss of cell adhesion and subsequent apoptosis. AKT2 (but not AKT1) ablation promoted cell cycle arrest at G0/G1, associated with downregulation of cyclin D, CDK6 and CDK2, and upregulation and cytoplasmic-to-nuclear redistribution of p27. The increase of p27 protein levels was due to more gene transcription and an increase in protein stability. The increased stability of p27 was induced by delocalisation of Skp2 and a lower level of p27 phosphorylation at Thr187. AKT1 and AKT2 ablation inhibited and stimulated PC-3 cell migration, respectively. An AKT isoform-specific function could be associated with its subcellular localization. We found that AKT1 and AKT2 were mainly localised in the cytoplasm and nucleus, respectively. In androgen-sensitive cell line LNCaP, the ablation of AKT1 or AKT2 caused apoptosis but in androgen-independent LNCaP sublines, the effect of AKT1 ablation was lower; whereas no changes were observed after AKT2 ablation. Taken together, our data show that AKT1 and AKT2 have non-redundant roles in the regulation of PC-3 cell proliferation and migration. These could be explained by their subcellular localization and/or the specific regulation of downstream effectors. Furthermore, contribution of AKT isoforms to the progression of prostate cancer may change from an androgen-sensitive to a hormone-refractory stage. These findings may help design new targeted strategies for inhibiting AKT isoforms in prostate cancer.

Gliomas are the most common type of primary brain tumor. Although tremendous progress has been achieved in the recent years in the diagnosis and treatment, its molecular etiology remains unknown. In this regard, epigenetics represents a new approach to study the mechanisms that control gene expression and function without changing the sequence of the genome. In the present paper we describe the main findings about the alterations of cell signaling pathways in the most aggressive glioma in the adult population, namely, glioblastoma, in which epigenetic mechanisms and the emerging role of cancer stem cell play a crucial function in the development of new biomarkers for its detection and prognosis and the corresponding development of new pharmacological strategies.

Introduction Androgen deprivation (AD) therapy is the principle treatment for high-risk locally advanced or metastatic prostate cancer. A significant number of patients develop resistance to this therapy leading to castration resistant prostate cancer (CRPC). A proposed mechanism for CRPC progression imply androgen receptor (AR) activation in the absence of its natural ligand. Epigenetics modifications are crucial events in AR-mediated gene expression regulation. For this reason, the main objective of the present study was to determine the role of DNA methylation in the progression to CRPC. Material and methods We used the Infinium 450K BeadChip methylation array to compare the DNA methylation profile of the prostate cancer cell lines LNCaP, the canonical androgen-dependent prostate cancer cell line, and the LNCaP abl, an androgen-independent derivative of LNCaP cells. CLDN3 silencing of LNCaP cells were performed by siRNA. The expression data of these cell lines were downloaded from GEO (GSE11428). Chromatin immunoprecipitation (ChIP) assay was performed with antibodies for AcH3, H3K4me3 and H3K27me3. Results and discussions The comparison of the data from the methylation array and the expression array provided a list of genes with deregulated expression in LNCaP abl cells by changes in promoter methylation: EGF, ELF5 and HYI were upregulated by promoter hypomethylation, and CLDN3, MARKS and THBS1 were downregulated by promoter hypermethylation among others. We selected CLDN3 for further study because codify for a protein implicated in tight junctions between cells. The loss of expression of CLDN3 in LNCaP abl cells were also related with the presence of the repressive histone mark H3K27me3 at the promoter region while in LNCaP cells, that express CLDN3, the active histone marks AcH3 and H3K4me3 were present. Finally, we wanted to analyse the functional relevance of the expression changes of CLDN3 in our cellular system. We observed an increase of the invasion in LNCaP abl cells. To verify that this phenotype is due, at least in part, to the loss of CLDN3, we inhibited its expression in LNCaP cells by siRNA and observed that the lack of CLDN3 expression increases the invasion recapitulating the phenotype of the abl cells. Conclusion We have identified a panel of genes deregulated in prostate cancer cell lines resistant to hormonal treatment by changes in DNA promoter methylation. In particular the epigenetic silencing of CLDN3 may have a relevant role in the development of CRPC.

To analyze, in older patients with schizophrenia, the methylation status of a set of genes associated with the pathophysiology of the disorder but including anatomical, clinical, and cognitive criteria in the experimental design that, in conjunction with the epigenetic status of specific genes, allows us to derive an integrative model.This study included 29 human brain samples from older schizophrenic patients with severe and mild cognitive impairment. We administered a comprehensive battery of neurocognitive tests to determine the size of the impairment across different cognitive domains. We focused our study on the analysis of the methylation pattern of 19 genes of major neurotransmitter systems using methylation-specific PCR and bisulfite genomic sequencing.Our results highlight an absence of hypermethylation and hypomethylation in older patients with schizophrenia and in healthy controls, irrespective of the degree of the cognitive deficit measured in the neuropsychological assessment (Fisher's exact test; p0.05).mRNA or protein expression level differences in genes of major neurotransmitter systems that are known to be altered in schizophrenia must be because of regulatory mechanisms other than the DNA methylation of its promoter regions, although our results highlight the idea that the analysis of the epigenetic mechanisms involved in schizophrenia represents a new approach that has the possibility of uncovering molecular mechanisms of dysregulated gene expression in this complex disorder.