Machine learning enables pan-cancer identification of mutational hotspots at persistent CTCF binding sites.

CCCTC-binding factor (CTCF) is an insulator protein that binds to a highly conserved DNA motif and facilitates regulation of three-dimensional (3D) nuclear architecture and transcription. CTCF binding sites (CTCF-BSs) reside in non-coding DNA and are frequently mutated in cancer. Our previous study identified a small subclass of CTCF-BSs that are resistant to CTCF knock down, termed persistent CTCF binding sites (P-CTCF-BSs). P-CTCF-BSs show high binding conservation and potentially regulate cell-type constitutive 3D chromatin architecture. Here, using ICGC sequencing data we made the striking observation that P-CTCF-BSs display a highly elevated mutation rate in breast and prostate cancer when compared to all CTCF-BSs. To address whether P-CTCF-BS mutations are also enriched in other cell-types, we developed CTCF-INSITE-a tool utilising machine learning to predict persistence based on genetic and epigenetic features of experimentally-determined P-CTCF-BSs. Notably, predicted P-CTCF-BSs also show a significantly elevated mutational burden in all 12 cancer-types tested. Enrichment was even stronger for P-CTCF-BS mutations with predicted functional impact to CTCF binding and chromatin looping. Using in vitro binding assays we validated that P-CTCF-BS cancer mutations, predicted to be disruptive, indeed reduced CTCF binding. Together this study reveals a new subclass of cancer specific CTCF-BS DNA mutations and provides insights into their importance in genome organization in a pan-cancer setting.

Characterisation and reproducibility of the HumanMethylationEPIC v2.0 BeadChip for DNA methylation profiling.

BACKGROUND: The Illumina family of Infinium Methylation BeadChip microarrays has been widely used over the last 15 years for genome-wide DNA methylation profiling, including large-scale and population-based studies, due to their ease of use and cost effectiveness. Succeeding the popular HumanMethylationEPIC BeadChip (EPICv1), the recently released Infinium MethylationEPIC v2.0 BeadChip (EPICv2) claims to extend genomic coverage to more than 935,000 CpG sites. Here, we comprehensively characterise the reproducibility, reliability and annotation of the EPICv2 array, based on bioinformatic analysis of both manifest data and new EPICv2 data from diverse biological samples. RESULTS: We find a high degree of reproducibility with EPICv1, evidenced by comparable sensitivity and precision from empirical cross-platform comparison incorporating whole genome bisulphite sequencing (WGBS), and high correlation between technical sample replicates, including between samples with DNA input levels below the manufacturer's recommendation. We provide a full assessment of probe content, evaluating genomic distribution and changes from previous array versions. We characterise EPICv2's new feature of replicated probes and provide recommendations as to the superior probes. In silico analysis of probe sequences demonstrates that probe cross-hybridisation remains a significant problem in EPICv2. By mapping the off-target sites at single nucleotide resolution and comparing with WGBS we show empirical evidence for preferential off-target binding. CONCLUSIONS: Overall, we find EPICv2 a worthy successor to the previous Infinium methylation microarrays, however some technical issues remain. To support optimal EPICv2 data analysis we provide an expanded version of the EPICv2 manifest to aid researchers in understanding probe design, data processing, choosing appropriate probes for analysis and for integration with methylation datasets from previous versions of the Infinium Methylation BeadChip.

The potential of epigenetic therapy to target the 3D epigenome in endocrine-resistant breast cancer.

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer-promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer-promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth.

Memory of stochastic single-cell apoptotic signaling promotes chemoresistance in neuroblastoma.

Gene expression noise is known to promote stochastic drug resistance through the elevated expression of individual genes in rare cancer cells. However, we now demonstrate that chemoresistant neuroblastoma cells emerge at a much higher frequency when the influence of noise is integrated across multiple components of an apoptotic signaling network. Using a JNK activity biosensor with longitudinal high-content and in vivo intravital imaging, we identify a population of stochastic, JNK-impaired, chemoresistant cells that exist because of noise within this signaling network. Furthermore, we reveal that the memory of this initially random state is retained following chemotherapy treatment across a series of in vitro, in vivo, and patient models. Using matched PDX models established at diagnosis and relapse from individual patients, we show that HDAC inhibitor priming cannot erase the memory of this resistant state within relapsed neuroblastomas but improves response in the first-line setting by restoring drug-induced JNK activity within the chemoresistant population of treatment-naïve tumors.

Epigenetic Therapies and Biomarkers in Breast Cancer.

Epigenetic therapies remain a promising, but still not widely used, approach in the management of patients with cancer. To date, the efficacy and use of epigenetic therapies has been demonstrated primarily in the management of haematological malignancies, with limited supportive data in solid malignancies. The most studied epigenetic therapies in breast cancer are those that target DNA methylation and histone modification; however, none have been approved for routine clinical use. The majority of pre-clinical and clinical studies have focused on triple negative breast cancer (TNBC) and hormone-receptor positive breast cancer. Even though the use of epigenetic therapies alone in the treatment of breast cancer has not shown significant clinical benefit, these therapies show most promise in use in combinations with other treatments. With improving technologies available to study the epigenetic landscape in cancer, novel epigenetic alterations are increasingly being identified as potential biomarkers of response to conventional and epigenetic therapies. In this review, we describe epigenetic targets and potential epigenetic biomarkers in breast cancer, with a focus on clinical trials of epigenetic therapies. We describe alterations to the epigenetic landscape in breast cancer and in treatment resistance, highlighting mechanisms and potential targets for epigenetic therapies. We provide an updated review on epigenetic therapies in the pre-clinical and clinical setting in breast cancer, with a focus on potential real-world applications. Finally, we report on the potential value of epigenetic biomarkers in diagnosis, prognosis and prediction of response to therapy, to guide and inform the clinical management of breast cancer patients.

DNA methylation is required to maintain both DNA replication timing precision and 3D genome organization integrity.

DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation.

BRG1 knockdown inhibits proliferation through multiple cellular pathways in prostate cancer.

BACKGROUND: BRG1 (encoded by SMARCA4) is a catalytic component of the SWI/SNF chromatin remodelling complex, with key roles in modulating DNA accessibility. Dysregulation of BRG1 is observed, but functionally uncharacterised, in a wide range of malignancies. We have probed the functions of BRG1 on a background of prostate cancer to investigate how BRG1 controls gene expression programmes and cancer cell behaviour. RESULTS: Our investigation of SMARCA4 revealed that BRG1 is over-expressed in the majority of the 486 tumours from The Cancer Genome Atlas prostate cohort, as well as in a complementary panel of 21 prostate cell lines. Next, we utilised a temporal model of BRG1 depletion to investigate the molecular effects on global transcription programmes. Depleting BRG1 had no impact on alternative splicing and conferred only modest effect on global expression. However, of the transcriptional changes that occurred, most manifested as down-regulated expression. Deeper examination found the common thread linking down-regulated genes was involvement in proliferation, including several known to increase prostate cancer proliferation (KLK2, PCAT1 and VAV3). Interestingly, the promoters of genes driving proliferation were bound by BRG1 as well as the transcription factors, AR and FOXA1. We also noted that BRG1 depletion repressed genes involved in cell cycle progression and DNA replication, but intriguingly, these pathways operated independently of AR and FOXA1. In agreement with transcriptional changes, depleting BRG1 conferred G1 arrest. CONCLUSIONS: Our data have revealed that BRG1 promotes cell cycle progression and DNA replication, consistent with the increased cell proliferation associated with oncogenesis.

Epigenetic reprogramming at estrogen-receptor binding sites alters 3D chromatin landscape in endocrine-resistant breast cancer.

Endocrine therapy resistance frequently develops in estrogen receptor positive (ER+) breast cancer, but the underlying molecular mechanisms are largely unknown. Here, we show that 3-dimensional (3D) chromatin interactions both within and between topologically associating domains (TADs) frequently change in ER+ endocrine-resistant breast cancer cells and that the differential interactions are enriched for resistance-associated genetic variants at CTCF-bound anchors. Ectopic chromatin interactions are preferentially enriched at active enhancers and promoters and ER binding sites, and are associated with altered expression of ER-regulated genes, consistent with dynamic remodelling of ER pathways accompanying the development of endocrine resistance. We observe that loss of 3D chromatin interactions often occurs coincidently with hypermethylation and loss of ER binding. Alterations in active A and inactive B chromosomal compartments are also associated with decreased ER binding and atypical interactions and gene expression. Together, our results suggest that 3D epigenome remodelling is a key mechanism underlying endocrine resistance in ER+ breast cancer.

Constitutively bound CTCF sites maintain 3D chromatin architecture and long-range epigenetically regulated domains.

The architectural protein CTCF is a mediator of chromatin conformation, but how CTCF binding to DNA is orchestrated to maintain long-range gene expression is poorly understood. Here we perform RNAi knockdown to reduce CTCF levels and reveal a shared subset of CTCF-bound sites are robustly resistant to protein depletion. The 'persistent' CTCF sites are enriched at domain boundaries and chromatin loops constitutive to all cell types. CRISPR-Cas9 deletion of 2 persistent CTCF sites at the boundary between a long-range epigenetically active (LREA) and silenced (LRES) region, within the Kallikrein (KLK) locus, results in concordant activation of all 8 KLK genes within the LRES region. CTCF genome-wide depletion results in alteration in Topologically Associating Domain (TAD) structure, including the merging of TADs, whereas TAD boundaries are not altered where persistent sites are maintained. We propose that the subset of essential CTCF sites are involved in cell-type constitutive, higher order chromatin architecture.

Integrated epigenomic analysis stratifies chromatin remodellers into distinct functional groups.

BACKGROUND: ATP-dependent chromatin remodelling complexes are responsible for establishing and maintaining the positions of nucleosomes. Chromatin remodellers are targeted to chromatin by transcription factors and non-coding RNA to remodel the chromatin into functional states. However, the influence of chromatin remodelling on shaping the functional epigenome is not well understood. Moreover, chromatin remodellers have not been extensively explored as a collective group across two-dimensional and three-dimensional epigenomic layers. RESULTS: Here, we have integrated the genome-wide binding profiles of eight chromatin remodellers together with DNA methylation, nucleosome positioning, histone modification and Hi-C chromosomal contacts to reveal that chromatin remodellers can be stratified into two functional groups. Group 1 (BRG1, SNF2H, CHD3 and CHD4) has a clear preference for binding at 'actively marked' chromatin and Group 2 (BRM, INO80, SNF2L and CHD1) for 'repressively marked' chromatin. We find that histone modifications and chromatin architectural features, but not DNA methylation, stratify the remodellers into these functional groups. CONCLUSIONS: Our findings suggest that chromatin remodelling events are synchronous and that chromatin remodellers themselves should be considered simultaneously and not as individual entities in isolation or necessarily by structural similarity, as they are traditionally classified. Their coordinated function should be considered by preference for chromatin features in order to gain a more accurate and comprehensive picture of chromatin regulation.

The Assessment of Clinical Usage and Prognostic Value of YKL-40 Serum Levels in Patients With Rectal Cancer Without Distant Metastasis.

BACKGROUND: Colorectal cancer is one of the most common and significant malignancies in the world. YKL-40 (chitinase-3-like protein 1) is involved in cell proliferation, migration, inflammation, and tissue remodeling; and serum levels of YKL-40 are associated with patient outcome in various cancers. The aim of this study was to assess the potential clinical usage of YKL-40 pretreatment serum levels as a prognostic biomarker in rectal cancer. METHODS: Concentrations of YKL-40 and standard tumor marker-Carcinoembryonic antigen (CEA)-were assessed in serum of 83 patients with rectal cancer without distant metastasis, and association with clinicopathological characteristics and disease-free and overall survival was evaluated. RESULTS: Concentration of YKL-40 was significantly higher in serum of patients with rectal cancer compared to healthy controls ( P = .0001), and YKL-40 levels were able to predict rectal cancer (area under the Receiver Operating Characteristic [ROC] curve = .769) with higher accuracy than CEA (area under the ROC curve = .728) in patients with early stage disease. Increased YKL-40 levels were significantly associated with age ( P = .001); however, no association with other clinicopathological characteristics was observed. Finally, in patients with recurrence, the percentage of cases with increased concentration of YKL-40 was significantly higher than in patients without recurrence ( P = .041), and Kaplan-Meier analysis demonstrated that elevated YKL-40 concentration is a predictor of poor overall survival in patients with rectal cancer. CONCLUSION: Pretreatment serum levels of YKL-40 may be a novel prognostic factor of overall and disease-free survival in patients with nonmetastatic colorectal cancer.

Disruption of the 3D cancer genome blueprint.

Recent advances in chromosome conformation capture technologies are improving the current appreciation of how 3D genome architecture affects its function in different cell types and disease. Long-range chromatin interactions are organized into topologically associated domains, which are known to play a role in constraining gene expression patterns. However, in cancer cells there are alterations in the 3D genome structure, which impacts on gene regulation. Disruption of topologically associated domains architecture can result in alterations in chromatin interactions that bring new regulatory elements and genes together, leading to altered expression of oncogenes and tumor suppressor genes. Here, we discuss the impact of genetic and epigenetic changes in cancer and how this affects the spatial organization of chromatin. Understanding how disruptions to the 3D architecture contribute to the cancer genome will provide novel insights into the principles of epigenetic gene regulation in cancer and mechanisms responsible for cancer associated mutations and rearrangements.

Three-dimensional disorganization of the cancer genome occurs coincident with long-range genetic and epigenetic alterations.

A three-dimensional chromatin state underpins the structural and functional basis of the genome by bringing regulatory elements and genes into close spatial proximity to ensure proper, cell-type-specific gene expression profiles. Here, we performed Hi-C chromosome conformation capture sequencing to investigate how three-dimensional chromatin organization is disrupted in the context of copy-number variation, long-range epigenetic remodeling, and atypical gene expression programs in prostate cancer. We find that cancer cells retain the ability to segment their genomes into megabase-sized topologically associated domains (TADs); however, these domains are generally smaller due to establishment of additional domain boundaries. Interestingly, a large proportion of the new cancer-specific domain boundaries occur at regions that display copy-number variation. Notably, a common deletion on 17p13.1 in prostate cancer spanning the TP53 tumor suppressor locus results in bifurcation of a single TAD into two distinct smaller TADs. Change in domain structure is also accompanied by novel cancer-specific chromatin interactions within the TADs that are enriched at regulatory elements such as enhancers, promoters, and insulators, and associated with alterations in gene expression. We also show that differential chromatin interactions across regulatory regions occur within long-range epigenetically activated or silenced regions of concordant gene activation or repression in prostate cancer. Finally, we present a novel visualization tool that enables integrated exploration of Hi-C interaction data, the transcriptome, and epigenome. This study provides new insights into the relationship between long-range epigenetic and genomic dysregulation and changes in higher-order chromatin interactions in cancer.

Alterations in Three-Dimensional Organization of the Cancer Genome and Epigenome.

The structural and functional basis of the genome is provided by the three-dimensional (3D) chromatin state. To enable accurate gene regulation, enhancer elements and promoter regions are brought into close spatial proximity to ensure proper, cell type-specific gene expression. In cancer, genetic and epigenetic processes can deregulate the transcriptional program. To investigate whether the 3D chromatin state is also disrupted in cancer we performed Hi-C chromosome conformation sequencing in normal and prostate cancer cells and compared the chromatin interaction maps with changes to the genome and epigenome. Notably, we find that additional topologically associated domain (TAD) boundaries are formed in cancer cells resulting in smaller TADs and altered gene expression profiles. The new TAD boundaries are commonly associated with copy-number changes observed in the cancer genome. We also identified new cancer-specific chromatin loops within TADs that are enriched for enhancers and promoters. Finally, we find that many of the long-range epigenetically silenced (LRES) and long-range epigenetically active (LREA) regions in cancer are characterized by differential chromatin interactions. Together our data provide a new insight into charting alterations in higher-order structure and the relationship with genetic, epigenetic, and transcriptional changes across the cancer genome.

Increased expression of miR-126 and miR-10a predict prolonged relapse-free time of primary oestrogen receptor-positive breast cancer following tamoxifen treatment.

BACKGROUND: Adjuvant tamoxifen is a valid treatment option for women with oestrogen receptor (ER)-positive breast cancer. However, up to 40% of patients experience distant or local recurrence or die. MicroRNAs have been suggested to be important prognosticators in breast cancer. This study aims to identify microRNAs with the potential to predict tamoxifen response. PATIENTS AND METHODS: We performed a global microRNA screen (1105 human microRNAs) in primary tumours of six matched pairs of postmenopausal, ER-positive breast cancer patients treated with tamoxifen, who were either recurrence free or had developed a recurrence (median follow up: 8.84 years; range: 1.28-12.7 years). Patients of this discovery set and the 81 patients of the validation set (median follow up: 8.64 years; range: 0.21-19.85 years) were treated at the Robert Bosch Hospital, Stuttgart, Germany, between 1986 and 2005. RESULTS: Out of the top 20 deregulated microRNAs (12 up-regulated, eight down-regulated) miR-126 (Hazard Ratio (HR) = 0.56, 95% confidence interval (CI): 0.38-0.83; Holm-adj. P = 0.022) and miR-10a (HR = 0.53, 95% CI: 0.33-0.85; Holm-adj. P = 0.031) were identified as significant predictors of tamoxifen outcome by multivariate Cox regression analysis in the independent validation set of 81 postmenopausal, ER-positive patients. Kaplan-Meier survival analyses based on cut-offs determined by receiver operating characteristics curves confirmed that a higher expression of miR-126 and miR-10a in the patients tumour was associated with longer relapse-free time (log-rank P = 0.037, P<0.0001, respectively). CONCLUSIONS: Our data suggest that miR-126 and miR-10a are independent predictors for tumour relapse in early postmenopausal breast cancer patients treated with adjuvant tamoxifen.