The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial ...inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies.
Non-coding RNA from pericentromeric satellite repeats are involved in stress-dependent splicing processes, maintenance of heterochromatin, and are required to protect genome stability. Here we show ...that the long non-coding satellite III RNA (SatIII) generates resistance against the topoisomerase IIa (TOP2A) inhibitor etoposide in lung cancer. Because heat shock conditions (HS) protect cells against the toxicity of etoposide, and SatIII is significantly induced under HS, we hypothesized that the protective effect could be traced back to SatIII. Using genome methylation profiles of patient-derived xenograft mouse models we show that the epigenetic modification of the SatIII DNA locus and the resulting SatIII expression predict chemotherapy resistance. In response to stress, SatIII recruits TOP2A to nuclear stress bodies, which protects TOP2A from a complex formation with etoposide and results in decreased DNA damage after treatment. We show that BRD4 inhibitors reduce the expression of SatIII, restoring etoposide sensitivity.
Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related deaths worldwide and is primarily treated with radiation, surgery, and platinum-based drugs like cisplatin and ...carboplatin. The major challenge in the treatment of NSCLC patients is intrinsic or acquired resistance to chemotherapy. Molecular markers predicting the outcome of the patients are urgently needed.
Here, we employed patient-derived xenografts (PDXs) to detect predictive methylation biomarkers for platin-based therapies. We used MeDIP-Seq to generate genome-wide DNA methylation profiles of 22 PDXs, their parental primary NSCLC, and their corresponding normal tissues and complemented the data with gene expression analyses of the same tissues. Candidate biomarkers were validated with quantitative methylation-specific PCRs (qMSP) in an independent cohort.
Comprehensive analyses revealed that differential methylation patterns are highly similar, enriched in PDXs and lung tumor-specific when comparing differences in methylation between PDXs versus primary NSCLC. We identified a set of 40 candidate regions with methylation correlated to carboplatin response and corresponding inverse gene expression pattern even before therapy. This analysis led to the identification of a promoter CpG island methylation of LDL receptor-related protein 12 (LRP12) associated with increased resistance to carboplatin. Validation in an independent patient cohort (n = 35) confirmed that LRP12 methylation status is predictive for therapeutic response of NSCLC patients to platin therapy with a sensitivity of 80% and a specificity of 84% (p < 0.01). Similarly, we find a shorter survival time for patients with LRP12 hypermethylation in the TCGA data set for NSCLC (lung adenocarcinoma).
Using an epigenome-wide sequencing approach, we find differential methylation patterns from primary lung cancer and PDX-derived cancers to be very similar, albeit with a lower degree of differential methylation in primary tumors. We identify LRP12 DNA methylation as a powerful predictive marker for carboplatin resistance. These findings outline a platform for the identification of epigenetic therapy resistance biomarkers based on PDX NSCLC models.
Background:
Methylation at 5 CpG sites was previously shown to classify chronic lymphocytic leukemia (CLL) into 3 prognostic subgroups. Here, we aimed to validate the marker set in an additional ...cohort and to evaluate its clinical utility for CLL patient stratification.
Methods:
We evaluated this epigenetic marker set in 79 German patients using bisulfite treatment followed by pyrosequencing and classification using a support vector machine-learning tool.
Results:
The n-CLL, i-CLL, and m-CLL classification was detected in 28 (35%), 10 (13%), and 41 (51%) patients, respectively. Epigenetic grouping was associated with IGHV mutational status (P = 2 × 10−12), isolated del13q (P = 9 × 10−6), del17p (P = .015), complex karyotype (P = .005), VH-usage, and clinical outcome as time to first treatment (P = 1.4 × 10−12) and overall survival (P = .003). Multivariate Cox regression analysis identified n-CLL as a factor for earlier treatment hazard ratio (HR), 6.3 (95% confidence interval CI 2.4-16.4; P = .0002) compared to IGHV mutational status (HR 4.6, 95% CI 1.9-11.3, P = .0008). In addition, when comparing the prognostic value of the epigenetic classification system with the IGHV classification, epigenetic grouping performed better compared to IGHV mutational status using Kaplan-Meier estimation and allowed the identification of a third, intermediate (i-CLL) group. Thus, our study confirmed the prognostic value of the epigenetic marker set for patient stratification in routine clinical diagnostics.
Mismatch repair (MMR)-deficiency increases the risk of colorectal tumorigenesis. To determine whether the tumors develop on a normal or disturbed epigenetic background and how radiation affects this, ...we quantified genome-wide histone H3 methylation profiles in macroscopic normal intestinal tissue of young radiated and untreated MMR-deficient VCMsh2
(Msh2
) mice months before tumor onset.
Histone H3 methylation increases in Msh2
compared to control Msh2
mice. Activating H3K4me3 and H3K36me3 histone marks frequently accumulate at genes that are H3K27me3 or H3K4me3 modified in Msh2
mice, respectively. The genes recruiting H3K36me3 enrich in gene sets associated with DNA repair, RNA processing, and ribosome biogenesis that become transcriptionally upregulated in the developing tumors. A similar epigenetic effect is present in Msh2
mice 4 weeks after a single-radiation hit, whereas radiation of Msh2
mice left their histone methylation profiles almost unchanged.
MMR deficiency results in genome-wide changes in histone H3 methylation profiles preceding tumor development. Similar changes constitute a persistent epigenetic signature of radiation-induced DNA damage.
Remodeling transcription by targeting bromodomain and extraterminal (BET) proteins has emerged as promising anticancer strategy. Here, we identify a novel synergistic interaction of the BET inhibitor ...JQ1 with the PI3Kα-specific inhibitor BYL719 to trigger mitochondrial apoptosis and to suppress tumor growth in models of rhabdomyosarcoma (RMS). RNA-Seq revealed that JQ1/BYL719 co-treatment shifts the overall balance of BCL-2 family gene expression towards apoptosis and upregulates expression of BMF, BCL2L11 (BIM), and PMAIP1 (NOXA) while downregulating BCL2L1 (BCL-x
). These changes were confirmed by qRT-PCR and western blot analysis. Ingenuity pathway analysis (IPA) of RNA-Seq data followed by validation qRT-PCR and western blot identified MYC and FOXO3a as potential transcription factors (TFs) upstream of the observed gene expression pattern. Immunoprecipitation (IP) studies showed that JQ1/BYL719-stimulated increase in BIM expression enhances the neutralization of antiapoptotic BCL-2, BCL-x
, and MCL-1. This promotes the activation of BAK and BAX and caspase-dependent apoptosis, as (1) individual silencing of BMF, BIM, NOXA, BAK, or BAX, (2) overexpression of BCL-2 or MCL-1 or (3) the caspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk) all rescue JQ1/BYL719-induced cell death. In conclusion, co-inhibition of BET proteins and PI3Kα cooperatively induces mitochondrial apoptosis by proapoptotic re-balancing of BCL-2 family proteins. This discovery opens exciting perspectives for therapeutic exploitation of BET inhibitors in RMS.
We report programmable receptors for the imaging‐based analysis of 5‐methylcytosine (5mC) in user‐defined DNA sequences of single cells. Using fluorescent transcription‐activator‐like effectors ...(TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co‐stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation in situ with nucleotide, locus, and cell resolution.
It needs two TALEs to tango: Programmable receptors for the imaging‐based analysis of 5‐methylcytosine (5mC) in DNA sequences of single cells are reported. Staining of SATIII DNA with pairs of 5mC‐sensitive and 5mC‐insensitive transcription‐activator‐like effectors (TALEs) allows the analysis of 5mC at a target nucleotide position and its direct correlation with the recruitment of transcription factor HSF1 as the initial step of nuclear stress body formation.
Remodeling transcription by targeting bromodomain and extraterminal (BET) proteins has emerged as promising anticancer strategy. Here, we identify a novel synergistic interaction of the BET inhibitor ...JQ1 with the PI3Kalpha-specific inhibitor BYL719 to trigger mitochondrial apoptosis and to suppress tumor growth in models of rhabdomyosarcoma (RMS). RNA-Seq revealed that JQ1/BYL719 co-treatment shifts the overall balance of BCL-2 family gene expression towards apoptosis and upregulates expression of BMF, BCL2L11 (BIM), and PMAIP1 (NOXA) while downregulating BCL2L1 (BCL-x.sub.L). These changes were confirmed by qRT-PCR and western blot analysis. Ingenuity pathway analysis (IPA) of RNA-Seq data followed by validation qRT-PCR and western blot identified MYC and FOXO3a as potential transcription factors (TFs) upstream of the observed gene expression pattern. Immunoprecipitation (IP) studies showed that JQ1/BYL719-stimulated increase in BIM expression enhances the neutralization of antiapoptotic BCL-2, BCL-x.sub.L, and MCL-1. This promotes the activation of BAK and BAX and caspase-dependent apoptosis, as (1) individual silencing of BMF, BIM, NOXA, BAK, or BAX, (2) overexpression of BCL-2 or MCL-1 or (3) the caspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk) all rescue JQ1/BYL719-induced cell death. In conclusion, co-inhibition of BET proteins and PI3Kalpha cooperatively induces mitochondrial apoptosis by proapoptotic re-balancing of BCL-2 family proteins. This discovery opens exciting perspectives for therapeutic exploitation of BET inhibitors in RMS.
Current molecular tumor diagnostics encompass panel sequencing to detect mutations, copy number alterations, and rearrangements. However, tumor suppressor genes can also be inactivated by methylation ...within their promoter region. These epigenetic alterations are so far rarely assessed in the clinical setting. Therefore, we established the AllCap protocol facilitating the combined detection of mutations and DNA methylation at the coding and promoter regions of 342 DNA repair genes in one experiment. We demonstrate the use of the protocol by applying it to ovarian cancer cell lines with different responsiveness to poly(ADP-ribose) polymerase inhibition. BRCA1, ATM, ATR, and EP300 mutations and methylation of the BRCA1 promoter were detected as potential predictors for therapy response. The required amount of input DNA was optimized, and the application to formalin-fixed, paraffin-embedded tissue samples was verified to improve the clinical applicability. Thus, by adding DNA methylation values to panel resequencings, the AllCap assay will add another important level of information to clinical tests and will improve stratification of patients for systemic therapies.
Genomics and epigenomics of colorectal cancer Schweiger, Michal-Ruth; Hussong, Michelle; Röhr, Christina ...
Wiley interdisciplinary reviews. Systems biology and medicine,
03/2013, Letnik:
5, Številka:
2
Journal Article
Recenzirano
Colorectal cancer is one of the most common cancer types worldwide and accounts for approximately 600,000 deaths annually. Work over the last decades has uncovered a number of tumor‐suppressor and ...oncogenes which are frequently mutated and might thus be responsible for the malignant transformation. However, only with the development of new high‐throughput technologies systematic analyses of the genome and epigenomes became feasible. While data generation has increased exponential, we are now faced with new challenges to transform these data into useful models that help predicting the outcome of genomic aberrations and to develop novel diagnostic and therapeutic strategies. As a basis for the modeling it is essential to understand and integrate current knowledge. We review previous and current ideas in colorectal cancer development and focus on a pathway oriented view. We show that colorectal cancer is a multilayer complex disease affecting the genome as well as the epigenome with direct consequences on the gene and microRNA (miRNA) expression signatures. The goal is to illustrate the current principles of colorectal cancer pathogenesis and to illustrate the need for elaborate computer modeling systems. WIREs Syst Biol Med 2013, 5:205–219. doi: 10.1002/wsbm.1206
This article is categorized under:
Analytical and Computational Methods > Analytical Methods
Laboratory Methods and Technologies > Genetic/Genomic Methods
Translational, Genomic, and Systems Medicine > Therapeutic Methods
Translational, Genomic, and Systems Medicine > Translational Medicine
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