27 results found
Beetch M, Lubecka K, Shen K, et al., 2019, Stilbenoid-Mediated Epigenetic Activation of Semaphorin 3A in Breast Cancer Cells Involves Changes in Dynamic Interactions of DNA with DNMT3A and NF1C Transcription Factor., Mol Nutr Food Res, Pages: e1801386-e1801386
SCOPE: Loci-specific increase in DNA methylation occurs in cancer and may underlie gene silencing. It is investigated whether dietary stilbenoids, resveratrol, and pterostilbene exert time-dependent effects on DNA methylation patterns and specifically methylation-silenced tumor suppressor genes in breast cancer cells. METHODS AND RESULTS: Following genome-wide DNA methylation analysis with Illumina-450K, changes characteristic of early and late response to stilbenoids are identified. Interestingly, often the same genes but at different CpG loci, the same gene families, or the same functional gene categories are affected. CpG loci that lose methylation in exposed cells correspond to genes functionally associated with cancer suppression. There is a group of genes, including SEMA3A, at which the magnitude of hypomethylation in response to stilbenoids rises with increasing invasive potential of cancer cells. Decreased DNA methylation at SEMA3A promoter and concomitant gene upregulation coincide with increased occupancy of active histone marks. Open chromatin upon exposure to stilbenoids may be linked to decreased DNMT3A binding followed by increased NF1C transcription factor occupancy. Sequestration of DNMT3A is possibly a result of stilbenoid-mediated increase in SALL3 expression, which was previously shown to bind and inhibit DNMT3A activity. CONCLUSIONS: The findings define mechanistic players in stilbenoid-mediated epigenetic reactivation of genes suppressing cancer.
Oltra SS, Pena-Chilet M, Vidal-Tomas V, et al., 2018, Methylation deregulation of miRNA promoters identifies miR124-2 as a survival biomarker in Breast Cancer in very young women, Scientific Reports, Vol: 8, ISSN: 2045-2322
MiRNAs are part of the epigenetic machinery, and are also epigenetically modified by DNA methylation. MiRNAs regulate expression of different genes, so any alteration in their methylation status may affect their expression. We aimed to identify methylation differences in miRNA encoding genes in breast cancer affecting women under 35 years old (BCVY), in order to identify potential biomarkers in these patients. In Illumina Infinium MethylationEPIC BeadChip samples (metEPICVal), we analysed the methylation of 9,961 CpG site regulators of miRNA-encoding genes present in the array. We identified 193 differentially methylated CpG sites in BCVY (p-value < 0.05 and methylation differences ±0.1) that regulated 83 unique miRNA encoding genes. We validated 10 CpG sites using two independent datasets based on Infinium Human Methylation 450k array. We tested gene expression of miRNAs with differential methylation in BCVY in a meta-analysis using The Cancer Genome Atlas (TCGA), Clariom D and Affymetrix datasets. Five miRNAs (miR-9, miR-124-2, miR-184, miR-551b and miR-196a-1) were differently expressed (FDR p-value < 0.01). Finally, only miR-124-2 shows a significantly different gene expression by quantitative real-time PCR. MiR-124-hypomethylation presents significantly better survival rates for older patients as opposed to the worse prognosis observed in BCVY, identifying it as a potential specific survival biomarker in BCVY.
Lubecka K, Flower K, Beetch M, et al., 2018, Loci-specific differences in blood DNA methylation in HBV-negative populations at risk for hepatocellular carcinoma development, Epigenetics, Vol: 13, Pages: 605-626, ISSN: 1559-2294
Late onset of clinical symptoms in hepatocellular carcinoma (HCC) results in late diagnosis and poor disease outcome. Approximately 85% of individuals with HCC have underlying liver cirrhosis. However, not all cirrhotic patients develop cancer. Reliable tools that would distinguish cirrhotic patients who will develop cancer from those who will not are urgently needed. We used the Illumina HumanMethylation450 BeadChip microarray to test whether white blood cell DNA, an easily accessible source of DNA, exhibits site-specific changes in DNA methylation in blood of diagnosed HCC patients (post-diagnostic, 24 cases, 24 controls) and in prospectively collected blood specimens of HCC patients who were cancer-free at blood collection (pre-diagnostic, 21 cases, 21 controls). Out of 22 differentially methylated loci selected for validation by pyrosequencing, 19 loci with neighbouring CpG sites (probes) were confirmed in the pre-diagnostic study group and subjected to verification in a prospective cirrhotic cohort (13 cases, 23 controls). We established for the first time 9 probes that could distinguish HBV-negative cirrhotic patients who subsequently developed HCC from those who stayed cancer-free. These probes were identified within regulatory regions of BARD1, MAGEB3, BRUNOL5, FXYD6, TET1, TSPAN5, DPPA5, KIAA1210, and LSP1. Methylation levels within DPPA5, KIAA1210, and LSP1 were higher in prospective samples from HCC cases versus cirrhotic controls. The remaining probes were hypomethylated in cases compared with controls. Using blood as a minimally invasive material and pyrosequencing as a straightforward quantitative method, the established probes have potential to be developed into a routine clinical test after validation in larger cohorts.
Sanchis S, Chilet MP, Martinez M, et al., 2017, Epigenomic landscape of breast cancer in very young women, 42nd Annual Meeting of the European-Society-of-Medical-Oncology (ESMO), Publisher: OXFORD UNIV PRESS, ISSN: 0923-7534
Lubecka K, Beetch M, Qiu J, et al., 2017, Loci-specific differences in blood DNA methylation for early detection of hepatocellular carcinoma, Annual Meeting of the American-Association-for-Cancer-Research (AACR), Publisher: AMER ASSOC CANCER RESEARCH, ISSN: 0008-5472
Beetch M, Lubecka K, Kurzava L, et al., 2017, Polyphenol-mediated epigenetic reactivation of tumor suppressor gene SEMA3A in breast cancer cells, Annual Meeting of the American-Association-for-Cancer-Research (AACR), Publisher: AMER ASSOC CANCER RESEARCH, ISSN: 0008-5472
Flanagan JM, Wilson A, Koo C, et al., 2017, Platinum-based chemotherapy induces methylation changes in blood DNA associated with overall survival in ovarian cancer patients, Clinical Cancer Research, Vol: 23, Pages: 2213-2222, ISSN: 1557-3265
PURPOSE: DNA damage repair can lead to epigenetic changes. DNA mismatch repair proteins bind to platinum DNA adducts and at sites of DNA damage can recruit the DNA methylating enzyme DNMT1, resulting in aberrant methylation. We hypothesised that DNA damage repair during platinum-based chemotherapy may cause aberrant DNA methylation in normal tissues of patients such as blood. EXPERIMENTAL DESIGN: We used Illumina 450k methylation arrays and bisulphite pyrosequencing to investigate methylation at presentation and relapse in blood DNA from ovarian cancer patients enrolled in the SCOTROC1 trial (n=247) and in a cohort of ovarian tumour DNA samples collected at first relapse (n=46). We used an ovarian cancer cell line model to investigate the role of the DNA mismatch repair gene MLH1 in platinum induced methylation changes. RESULTS: Specific CpG methylation changes in blood at relapse are observed following platinum-based chemotherapy and are associated with patient survival, independent of other clinical factors (HR=3.7; 95%CI 1.8-7.6, p=2.8x10-4). Similar changes occur in ovarian tumours at relapse, also associate with patient survival (HR=2.6; 95%CI 1.0-6.8, p=0.048). Using an ovarian cancer cell line model, we demonstrate that functional mismatch repair (MMR) increases the frequency of platinum-induced methylation. CONCLUSION: DNA methylation in blood at relapse following chemotherapy, and not at presentation, is informative about ovarian cancer patient survival. Functional DNA mismatch repair increases the frequency of DNA methylation changes induced by platinum. DNA methylation in blood following chemotherapy could provide a non-invasive means of monitoring patients' epigenetic responses to treatment without requiring a tumour biopsy.
Stefanska B, Kurzava L, Lubecka K, et al., 2017, Epigenetic Regulation of WNT and Hedgehog Oncogenic Signaling in Breast Cancer Cells in Response to Dietary Polyphenols, Experimental Biology (EB) Annual Meeting, Publisher: WILEY, ISSN: 0892-6638
Phelps DL, Borley J, Flower K, et al., 2017, Methylation of MYLK3 gene promoter region: a biomarker to stratify surgical care in ovarian cancer in a multi-centre study, British Journal of Cancer, Vol: 116, Pages: 1287-1293, ISSN: 1532-1827
BackgroundSurvival benefit from surgical debulking of ovarian cancer (OC) is well established but some women, despite total macroscopic clearance of disease, still have poor prognosis. We aimed to identify biomarkers to predict benefit from conventional surgery.MethodsClinical data from women debulked for high-stage OC was analysed (Hammersmith Hospital, London, UK; 2001-2014). Infinium’s HumanMethylation27 array interrogated tumour-DNA for differentially-methylated CpG sites, correlated to survival, in patients with the least residual disease (RD) (Hammersmith Array). Validation was performed using bisulphite pyrosequencing (Charité Hospital, Berlin, Germany cohort) and The Cancer Genome Atlas’ (TCGA) methylation dataset. Kaplan-Meier curves and Cox models tested survival.ResultsAltogether 803 women with serous ovarian cancer were studied. No RD was associated with significantly improved overall- (OS) (hazard ratio [HR] 1.25, 95% CI 1.06-1.47; P=0.0076) and progression-free survival (PFS) (HR 1.23, 1.05-1.43; P=0.012) (Hammersmith database n=430). Differentially-methylated loci within FGF4, FGF21, MYLK2, MYLK3, MYL7, and ITGAE associated with survival. Patients with the least RD had significantly better OS with higher methylation of MYLK3 (Hammersmith (HR 0.51, 0.31-0.84; P=0.01), Charité (0.46, 0.21-1.01; P=0.05), TCGA (0.64, 0.44-0.93; P=0.02)). ConclusionMYLK3 methylation is associated with improved OS in patients with the least RD, which could potentially be used to determine response to surgery.
Flower KJ, Ghaem-Maghami S, Brown R, 2017, Is there a role for epigenetic enhancement of immunomodulatory approaches to cancer treatment?, Current Cancer Drug Targets, Vol: 17, ISSN: 1873-5576
The efficacy of cancer immunotherapy relies on the ability of the host immune system to recognise the cancer as non-self and eliminate it from the body. Whilst this is an extremely fertile area of medical research, with positive clinical trials showing durable responses, attention must be paid to the subset of patients that do not respond to these treatments. Immune surveillance and immunoediting by the host could itself select for immune-evasive tumour cells during tumour development leading to immunotherapy resistance. One such mechanism of non-efficacy or resistance is the epigenetic silencing of a specific gene required in the immunotherapy response pathway. Epigenetics is the study of the control of expression patterns in a cell via mechanisms not involving a change in DNA sequence. All tumour types show aberrant epigenetic regulation of genes involved in all the hallmarks of cancer, including immunomodulation. Inhibition of key enzymes involved in maintenance of epigenetic states is another important area of research for new treatment strategies for cancer. Could epigenetic therapies be used to successfully enhance the action of immunomodulatory agents in cancer, and are they acting in the way we imagine? An understanding of the effects of epigenetic therapies on immunological pathways in both the tumour and host cells, especially the tumour microenvironment, will be essential to further develop such combination approaches.
Flanagan JM, Curry E, Stirling L, et al., 2017, Epigenome-wide association study for breast cancer risk using whole genome and target captured bisulphite sequencing: A pooled case-control study nested in the breakthrough generations study, San Antonio Breast Cancer Symposium, Publisher: AMER ASSOC CANCER RESEARCH, ISSN: 0008-5472
Whilding LM, Parente-Pereira AC, Zabinski T, et al., 2017, Targeting of Aberrant alpha v beta 6 Integrin Expression in Solid Tumors Using Chimeric Antigen Receptor-Engineered T Cells, Molecular Therapy, Vol: 25, Pages: 259-273, ISSN: 1525-0016
Expression of the αvβ6 integrin is upregulated in several solid tumors. In contrast, physiologic expression of this epithelial-specific integrin is restricted to development and epithelial re-modeling. Here, we describe, for the first time, the development of a chimeric antigen receptor (CAR) that couples the recognition of this integrin to the delivery of potent therapeutic activity in a diverse repertoire of solid tumor models. Highly selective targeting αvβ6 was achieved using a foot and mouth disease virus-derived A20 peptide, coupled to a fused CD28+CD3 endodomain. To achieve selective expansion of CAR T cells ex vivo, an IL-4-responsive fusion gene (4αβ) was co-expressed, which delivers a selective mitogenic signal to engineered T cells only. In vivo efficacy was demonstrated in mice with established ovarian, breast, and pancreatic tumor xenografts, all of which express αvβ6 at intermediate to high levels. SCID beige mice were used for these studies because they are susceptible to cytokine release syndrome, unlike more immune-compromised strains. Nonetheless, although the CAR also engages mouse αvβ6, mild and reversible toxicity was only observed when supra-therapeutic doses of CAR T cells were administered parenterally. These data support the clinical evaluation of αvβ6 re-targeted CAR T cell immunotherapy in solid tumors that express this integrin.
Chatterjee J, Dai W, Abd Aziz NH, et al., 2016, Clinical use of programmed cell death-1 (PD-1) and its ligand (PD-L1) expression as discriminatory and predictive markers in ovarian cancer, Clinical Cancer Research, Vol: 23, Pages: 3453-3460, ISSN: 1557-3265
Purpose We aimed to establish whether PD-1 and PD-L1 expression, in ovarian cancer (OC) tumour tissue and blood, could be used as biomarkers for discrimination of tumour histology and prognosis of OC. Experimental Design Immune cells were separated from blood, ascites and tumour tissue obtained from women with suspected OC and studied for the differential expression of possible immune biomarkers using flow cytometry. PD-L1 expression on tumour associated inflammatory cells was assessed by immunohistochemistry and tissue microarray. Plasma soluble PD-L1 was measured using sandwich ELISA. The relationships among immune markers were explored using hierarchical cluster analyses. Results Biomarkers from the discovery cohort that associated with PD-L1+ cells were found. PD-L1+ CD14+ cells and PD-L1+ CD11c+ cells in the monocyte gate showed a distinct expression pattern when comparing benign tumours and epithelial ovarian cancers (EOC) - confirmed in the validation cohort. Receiver operating characteristic curves showed PD-L1+ and PD-L1+ CD14+ cells in the monocyte gate performed better than the well-established tumour marker CA-125 alone. Plasma soluble PD-L1 was elevated in EOC patients compared to healthy women and patients with benign ovarian tumours. Low total PD-1+ expression on lymphocytes was associated with improved survival. Conclusions Differential expression of immunological markers relating to the PD-1/PD-L1 pathway in blood can be used as potential diagnostic and prognostic markers in EOC. These data have implications for the development and trial of anti PD-1/PD-L1 therapy in ovarian cancer.
Lubecka K, Kurzava L, Flower K, et al., 2016, Differences in gene-specific DNA methylation in blood DNA as a biomarker for early detection of hepatocellular carcinoma in populations at risk, AACR 107th Annual Meeting on Bioinformatics and Systems Biology, Publisher: AMER ASSOC CANCER RESEARCH, ISSN: 0008-5472
Lubecka K, Kurzava L, Flower K, et al., 2016, Stilbenoids remodel the DNA methylation patterns in breast cancer cells and inhibit oncogenic NOTCH signaling through epigenetic regulation of MAML2 transcriptional activity, Carcinogenesis, Vol: 37, Pages: 656-668, ISSN: 1460-2180
DNA hypomethylation was previously implicated in cancer progression and metastasis. The purpose of this study was to examine whether stilbenoids, resveratrol and pterostilbene thought to exert anticancer effects, target genes with oncogenic function for de novo methylation and silencing, leading to inactivation of related signaling pathways. Following Illumina 450K, genome-wide DNA methylation analysis reveals that stilbenoids alter DNA methylation patterns in breast cancer cells. On average, 75% of differentially methylated genes have increased methylation, and these genes are enriched for oncogenic functions, including NOTCH signaling pathway. MAML2, a coactivator of NOTCH targets, is methylated at the enhancer region and transcriptionally silenced in response to stilbenoids, possibly explaining the downregulation of NOTCH target genes. The increased DNA methylation at MAML2 enhancer coincides with increased occupancy of repressive histone marks and decrease in activating marks. This condensed chromatin structure is associated with binding of DNMT3B and decreased occupancy of OCT1 transcription factor at MAML2 enhancer, suggesting a role of DNMT3B in increasing methylation of MAML2 after stilbenoid treatment. Our results deliver a novel insight into epigenetic regulation of oncogenic signals in cancer and provide support for epigenetic-targeting strategies as an effective anticancer approach.
Flower KJ, Shenker NS, el-Bahrawy M, et al., 2016, DNA methylation profiling to assess pathogenicity of BRCA1 unclassified variants in breast cancer., Epigenetics, Vol: 10, Pages: 1121-1132, ISSN: 1559-2308
Germline pathogenic mutations in BRCA1 increase risk of developing breast cancer. Screening for mutations in BRCA1 frequently identifies sequence variants of unknown pathogenicity and recent work has aimed to develop methods for determining pathogenicity. We previously observed that tumor DNA methylation can differentiate BRCA1-mutated from BRCA1-wild type tumors. We hypothesized that we could predict pathogenicity of variants based on DNA methylation profiles of tumors that had arisen in carriers of unclassified variants. We selected 150 FFPE breast tumor DNA samples [47 BRCA1 pathogenic mutation carriers, 65 BRCAx (BRCA1-wild type), 38 BRCA1 test variants] and analyzed a subset (n=54) using the Illumina 450K methylation platform, using the remaining samples for bisulphite pyrosequencing validation. Three validated markers (BACH2, C8orf31, and LOC654342) were combined with sequence bioinformatics in a model to predict pathogenicity of 27 variants (independent test set). Predictions were compared with standard multifactorial likelihood analysis. Prediction was consistent for c.5194-12G>A (IVS 19-12 G>A) (P>0.99); 13 variants were considered not pathogenic or likely not pathogenic using both approaches. We conclude that tumor DNA methylation data alone has potential to be used in prediction of BRCA1 variant pathogenicity but is not independent of estrogen receptor status and grade, which are used in current multifactorial models to predict pathogenicity.
van veldhoven K, Polidoro S, Baglietto L, et al., 2015, Epigenome-wide association study reveals decreased average methylation levels years before breast cancer diagnosis, Clinical Epigenetics, Vol: 7, ISSN: 1868-7083
Background. Interest in the potential of DNA methylation in peripheral blood as a biomarker of cancer risk is increasing. We aimed to assess whether epigenome-wide DNA methylation measured in peripheral blood samples obtained before onset of the disease is associated with increased risk of breast cancer.Methods. We report on three independent prospective nested case-control studies from the European Prospective Investigation into Cancer and Nutrition (EPIC-Italy, n=162 matched case-control pairs); the Norwegian Women and Cancer study (NOWAC, n=168 matched pairs); and the Breakthrough Generations Study (BGS, n=548 matched pairs). We used the Illumina 450k array to measure methylation in the EPIC and NOWAC cohorts. Whole genome bisulphite sequencing (WGBS) was performed on the BGS cohort using pooled DNA samples, combined to reach 50x-coverage across ~16 million CpG sites in the genome including 450k array CpG sites. Mean β values over all probes were calculated as a measurement for epigenome-wide methylation.Results. In EPIC we found that high epigenome-wide methylation was associated with lower risk of breast cancer (OR per 1SD=0.61, 95%CI 0.47–0.80; -0.2% average difference in epigenome-wide methylation for cases and controls). Specifically, this was observed in gene bodies (OR=0.51, 95%CI 0.38–0.69) but not in gene promoters (OR=0.92, 95%CI 0.64–1.32). The association was not replicated in NOWAC (OR=1.03 95%CI 0.81–1.30). The reasons for heterogeneity across studies are unclear. However, data from the BGS cohort was consistent with epigenome-wide hypomethylation in breast cancer cases across the overlapping 450k probe sites (difference in average epigenome-wide methylation in case and control DNA pools=-0.2%).Conclusions. We conclude that epigenome-wide hypomethylation of DNA from pre-diagnostic blood samples may be predictive of breast cancer risk and may thus be useful as a clinical biomarker.
Shenker NS, Flower KJ, Wilhelm-Benartzi C, et al., 2015, Transcriptional implications of intragenic DNA methylation in the estrogen receptor alpha gene in breast cancer cells and tissues, 106th Annual Meeting of the American-Association-for-Cancer-Research (AACR), Publisher: American Association for Cancer Research, ISSN: 1538-7445
Lubecka-Pietruszewska K, Kurzava L, Buvala H, et al., 2015, Differential DNA methylation in peripheral blood DNA as a biomarker of hepatocellular carcinoma risk, 106th Annual Meeting of the American-Association-for-Cancer-Research (AACR), Publisher: AMER ASSOC CANCER RESEARCH, ISSN: 0008-5472
Shenker NS, Flower KJ, Wilhelm-Benartzi CS, et al., 2015, Transcriptional implications of intragenic DNA methylation in the oestrogen receptor alpha gene in breast cancer cells and tissues., BMC Cancer, Vol: 15, Pages: 337-337, ISSN: 1471-2407
BACKGROUND: DNA methylation variability regions (MVRs) across the oestrogen receptor alpha (ESR1) gene have been identified in peripheral blood cells from breast cancer patients and healthy individuals. In contrast to promoter methylation, gene body methylation may be important in maintaining active transcription. This study aimed to assess MVRs in ESR1 in breast cancer cell lines, tumour biopsies and exfoliated epithelial cells from expressed breast milk (EBM), to determine their significance for ESR1 transcription. METHODS: DNA methylation levels in eight MVRs across ESR1 were assessed by pyrosequencing bisulphite-converted DNA from three oestrogen receptor (ER)-positive and three ER-negative breast cancer cell lines. DNA methylation and expression were assessed following treatment with DAC (1 μM), or DMSO (controls). ESR1 methylation levels were also assayed in DNA from 155 invasive ductal carcinoma biopsies provided by the Breast Cancer Campaign Tissue Bank, and validated with DNA methylation profiles from the TCGA breast tumours (n = 356 ER-pos, n = 109 ER-neg). DNA methylation was profiled in exfoliated breast epithelial cells from EBM using the Illumina 450 K (n = 36) and pyrosequencing in a further 53 donor samples. ESR1 mRNA levels were measured by qRT-PCR. RESULTS: We show that ER-positive cell lines had unmethylated ESR1 promoter regions and highly methylated intragenic regions (median, 80.45%) while ER-negative cells had methylated promoters and lower intragenic methylation levels (median, 38.62%). DAC treatment increased ESR1 expression in ER-negative cells, but significantly reduced methylation and expression of ESR1 in ER-positive cells. The ESR1 promoter was unmethylated in breast tumour biopsies with high levels of intragenic methylation, independent of ER status. However, ESR1 methylation in the strongly ER-positive EBM DNA samples were very similar to ER-positive tumour cell lines. CONCLUSION:
Ramasubramanyan S, Kanhere A, Osborn K, et al., 2012, Genome-Wide Analyses of Zta Binding to the Epstein-Barr Virus Genome Reveals Interactions in both Early and Late Lytic Cycles and an Epigenetic Switch Leading to an Altered Binding Profile, JOURNAL OF VIROLOGY, Vol: 86, Pages: 12494-12502, ISSN: 0022-538X
Ramasubramanyan S, Osborn K, Flower K, et al., 2012, Dynamic Chromatin Environment of Key Lytic Cycle Regulatory Regions of the Epstein-Barr Virus Genome, JOURNAL OF VIROLOGY, Vol: 86, Pages: 1809-1819, ISSN: 0022-538X
Flower K, Thomas D, Heather J, et al., 2011, Epigenetic Control of Viral Life-Cycle by a DNA-Methylation Dependent Transcription Factor, PLOS ONE, Vol: 6, ISSN: 1932-6203
Flower K, Hellen E, Newport MJ, et al., 2010, Evaluation of a Prediction Protocol to Identify Potential Targets of Epigenetic Reprogramming by the Cancer Associated Epstein Barr Virus, PLOS ONE, Vol: 5, ISSN: 1932-6203
Heather J, Flower K, Isaac S, et al., 2009, The Epstein-Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1, JOURNAL OF GENERAL VIROLOGY, Vol: 90, Pages: 1450-1454, ISSN: 0022-1317
Schelcher C, Al Mehairi S, Verrall E, et al., 2007, Atypical bZIP domain of viral transcription factor contributes to stability of dimer formation and transcriptional function, JOURNAL OF VIROLOGY, Vol: 81, Pages: 7149-7155, ISSN: 0022-538X
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