[Show abstract][Hide abstract] ABSTRACT: Expression of oestrogen receptor (ESR1) determines whether a breast cancer patient receives endocrine therapy, but does not guarantee patient response. The molecular factors that define endocrine response in ESR1-positive breast cancer patients remain poorly understood. Here we characterize the DNA methylome of endocrine sensitivity and demonstrate the potential impact of differential DNA methylation on endocrine response in breast cancer. We show that DNA hypermethylation occurs predominantly at oestrogen-responsive enhancers and is associated with reduced ESR1 binding and decreased gene expression of key regulators of ESR1 activity, thus providing a novel mechanism by which endocrine response is abated in ESR1-positive breast cancers. Conversely, we delineate that ESR1-responsive enhancer hypomethylation is critical in transition from normal mammary epithelial cells to endocrine-responsive ESR1-positive cancer. Cumulatively, these novel insights highlight the potential of ESR1-responsive enhancer methylation to both predict ESR1-positive disease and stratify ESR1-positive breast cancer patients as responders to endocrine therapy.
[Show abstract][Hide abstract] ABSTRACT: Background
Dysregulation of the epigenome is a common event in malignancy; however, deciphering the earliest cancer-associated epigenetic events remains a challenge. Cancer epigenome studies to date have primarily utilised cancer cell lines or clinical samples, where it is difficult to identify the initial epigenetic lesions from those that occur over time. Here, we analysed the epigenome of human mammary epithelial cells (HMEC) and a matched variant cell population (vHMEC) that have spontaneously escaped senescence and undergone partial carcinogenic transformation. Using this model of basal-like breast carcinogenesis, we provide striking new insights into the very first epigenetic changes that occur during the initial stages of malignancy.
The first phase of malignancy is defined by coordinated changes in the epigenome. At the chromatin level, this is embodied in long-range epigenetic deregulation, which involves the concomitant but atypical acquisition or loss of active and repressive histone modifications across large regional blocks. Changes in DNA methylation also occurs in a highly coordinated manner. We identified differentially methylated regions (DMRs) in the very earliest passages of vHMECs. Notably, we find that differential methylation targets loci regulated by key transcription factors including p53, AHR and E2F family members suggesting that epigenetic deregulation of transcription factor binding is a key event in breast carcinogenesis. Interestingly, DMRs identified in vHMEC are extensively methylated in breast cancer, with hypermethylation frequently encroaching into neighbouring regions. A subset of vHMEC DMRs exhibited a strong basal-like cancer specific hypermethylation.
Here, we generated epigenome-wide maps of the earliest phase of breast malignancy and show long-range epigenetic deregulation and coordinated DNA hypermethylation targets loci regulated by key transcription factors. These findings support a model where induction of breast cancer occurs through epigenetic disruption of transcription factor binding leading to deregulation of cancer-associated transcriptional networks. With their stability and very early occurrence, vHMECs hypermethylated loci could serve as excellent biomarkers for the initial detection of basal breast cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s13148-015-0086-0) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel options for tumour stratification. Here, we perform whole-genome methylation capture sequencing on small amounts of DNA isolated from formalin-fixed, paraffin-embedded tissue from triple-negative breast cancer (TNBC) and matched normal samples. We identify differentially methylated regions (DMRs) enriched with promoters associated with transcription factor binding sites and DNA hypersensitive sites. Importantly, we stratify TNBCs into three distinct methylation clusters associated with better or worse prognosis and identify 17 DMRs that show a strong association with overall survival, including DMRs located in the Wilms tumour 1 (WT1) gene, bi-directional-promoter and antisense WT1-AS. Our data reveal that coordinated hypermethylation can occur in oestrogen receptor-negative disease, and that characterizing the epigenetic framework provides a potential signature to stratify TNBCs. Together, our findings demonstrate the feasibility of profiling the cancer methylome with limited archival tissue to identify regulatory regions associated with cancer.
[Show abstract][Hide abstract] ABSTRACT: One of the best-characterized oncogenic mechanisms in breast cancer is the aberrant activation of phosphatidylinositol-3-kinase, protein kinase B, and mammalian target of rapamycin signaling. In both endocrine-resistant disease and breast cancer stem cells, this is commonly caused by specific genetic lesions or amplification of key pathway components or both. These observations have generated two interesting hypotheses. Firstly, do these genetic anomalies provide clinically significant biomarkers predictive of endocrine resistance? Secondly, do tamoxifen-resistant breast cancer cells emerge from a stem-like cell population? New studies, published in Breast Cancer Research, raise the possibility that these hypotheses are intrinsically linked.
Breast Cancer Research 05/2014; 16(3):R48. DOI:10.1186/bcr3659 · 5.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acquired resistance to the anti-estrogen tamoxifen remains a significant challenge in breast cancer management. In this study we used an integrative approach to characterize global protein expression and tyrosine-phosphorylation events in tamoxifen-resistant MCF7 breast cancer cells (TamR) compared with parental controls. Quantitative mass-spectrometry and computational approaches were combined to identify perturbed signalling networks, and candidate regulatory proteins were functionally interrogated by siRNA-mediated knockdown. Network analysis revealed that cellular metabolism was perturbed in TamR cells, together with pathways enriched for proteins associated with growth-factor, cell-cell and cell-matrix-initiated signalling. Consistent with known roles for Ras/MAPK and PI3-kinase signalling in tamoxifen resistance, tyrosine phosphorylated MAPK1, SHC1 and PIK3R2 were elevated in TamR cells. Phosphorylation of the tyrosine kinase Yes and expression of the actin-binding protein MARCKS were elevated 2- and 8-fold in TamR cells respectively, and were selected for further analysis. Knockdown of either protein in TamR cells had no effect on anti-estrogen-sensitivity, but significantly decreased cell motility. MARCKS expression was significantly higher in breast cancer cell lines than normal mammary epithelial cells and in ER-negative versus ER-positive breast cancer cell lines. In primary breast cancers, cytoplasmic MARCKS staining was significantly higher in basal-like and HER2 cancers than in luminal cancers, and was independently predictive of poor survival in multivariate analyses of the whole cohort (p<0.0001) and in ER-positive patients (p=0.0005). These findings provide network-level insights into the molecular alterations associated with the tamoxifen-resistant phenotype and identify MARCKS as a potential biomarker of therapeutic responsiveness that may assist stratification of patients for optimal therapy. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Overexpression of the anti-apoptotic factor, BCL-2, is a frequent feature of malignant disease and is commonly associated with poor prognosis and resistance to conventional chemotherapy. In breast cancer, however, high BCL-2 expression is associated with favourable prognosis, estrogen receptor (ER) positivity and low tumour grade; whilst low expression is included in several molecular signatures associated with resistance to endocrine therapy. In the present study, we correlate BCL-2 expression and DNA methylation profiles in human breast cancer and in multiple cell models of acquired endocrine-resistance to determine whether BCL-2 hypermethylation could provide a useful biomarker of response to cytotoxic therapy. In human disease, diminished expression of BCL-2 was associated with hypermethylation of the second exon, in a region that overlapped a CpG island and an ER-binding site. Hypermethylation of this region, which occurred in 10% of primary tumours, provided a stronger predictor of patient survival (p=0.019) when compared to gene expression (n=522). In multiple cell-models of acquired endocrine-resistance, BCL-2 expression was significantly reduced in parallel with increased DNA methylation of the exon 2 region. The reduction of BCL-2 expression in endocrine-resistant cells lowered their apoptotic threshold to anti-mitotic agents: nocodazole, paclitaxel and the PLK1 inhibitor, BI2536. This phenomenon could be reversed with ectopic expression of BCL-2, and rescued with the BCL-2 inhibitor, ABT-737. Collectively, these data imply that BCL-2 hypermethylation provides a robust biomarker of response to current and next generation cytotoxic agents in endocrine-resistant breast cancer, which may prove beneficial in directing therapeutic strategy for patients with non-resectable, metastatic disease.
Molecular Cancer Therapeutics 07/2013; 12(9). DOI:10.1158/1535-7163.MCT-13-0012 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that during pregnancy the E-twenty-six (ETS) transcription factor ELF5 directs the differentiation of mammary progenitor cells toward the estrogen receptor (ER)-negative and milk producing cell lineage, raising the possibility that ELF5 may suppress the estrogen sensitivity of breast cancers. To test this we constructed inducible models of ELF5 expression in ER positive luminal breast cancer cells and interrogated them using transcript profiling and chromatin immunoprecipitation of DNA followed by DNA sequencing (ChIP-Seq). ELF5 suppressed ER and FOXA1 expression and broadly suppressed ER-driven patterns of gene expression including sets of genes distinguishing the luminal molecular subtype. Direct transcriptional targets of ELF5, which included FOXA1, EGFR, and MYC, accurately classified a large cohort of breast cancers into their intrinsic molecular subtypes, predicted ER status with high precision, and defined groups with differential prognosis. Knockdown of ELF5 in basal breast cancer cell lines suppressed basal patterns of gene expression and produced a shift in molecular subtype toward the claudin-low and normal-like groups. Luminal breast cancer cells that acquired resistance to the antiestrogen Tamoxifen showed greatly elevated levels of ELF5 and its transcriptional signature, and became dependent on ELF5 for proliferation, compared to the parental cells. Thus ELF5 provides a key transcriptional determinant of breast cancer molecular subtype by suppression of estrogen sensitivity in luminal breast cancer cells and promotion of basal characteristics in basal breast cancer cells, an action that may be utilised to acquire antiestrogen resistance.
[Show abstract][Hide abstract] ABSTRACT: In the present study, we have taken the novel approach of using an in vitro model representative of tamoxifen-withdrawal subsequent to clinical relapse to achieve a greater understanding of the mechanisms that serve to maintain the resistant-cell phenotype, independent of any agonistic impact of tamoxifen, to identify potential novel therapeutic approaches for this disease state. Following tamoxifen withdrawal, tamoxifen-resistant MCF-7 cells conserved both drug resistance and an increased basal rate of proliferation in an oestrogen deprived environment, despite reduced epidermal growth-factor receptor expression and reduced sensitivity to gefitinib challenge. Although tamoxifen-withdrawn cells retained ER expression, a sub-set of ER-responsive genes, including pS2 and progesterone receptor (PgR), were down-regulated by promoter DNA methylation, as confirmed by clonal bisulphite sequencing experiments. Following promoter demethylation with 5-Azacytidine (5-Aza), the co-addition of oestradiol (E2) restored gene expression in these cells. In addition, 5-Aza/E2 co-treatment induced a significant anti-proliferative effect in the tamoxifen-withdrawn cells, in-contrast to either agent used alone. Microarray analysis was undertaken to identify genes specifically up regulated by this co-treatment. Several anti-proliferative gene candidates were identified and their promoters were confirmed as more heavily methylated in the tamoxifen resistant vs sensitive cells. One such gene candidate, growth differentiation factor 15 (GDF15), was carried forward for functional analysis. The addition of 5-Aza/E2 was sufficient to de-methylate and activate GDF15 expression in the tamoxifen resistant cell-lines, whilst in parallel, treatment with recombinant GDF15 protein decreased cell survival. These data provide evidence to support a novel concept that long-term tamoxifen exposure induces epigenetic silencing of a cohort of oestrogen-responsive genes whose function is associated with negative proliferation control. Furthermore, reactivation of such genes using epigenetic drugs could provide a potential therapeutic avenue for the management of tamoxifen-resistant breast cancer.
PLoS ONE 07/2012; 7(7):e40466. DOI:10.1371/journal.pone.0040466 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cyclin E2, but not cyclin E1, is included in several gene signatures that predict disease progression in either tamoxifen-resistant or metastatic breast cancer. We therefore examined the role of cyclin E2 in antiestrogen resistance in vitro and its potential for therapeutic targeting through cyclin-dependent kinase (CDK) inhibition. High expression of CCNE2, but not CCNE1, was characteristic of the luminal B and HER2 subtypes of breast cancer and was strongly predictive of shorter distant metastasis-free survival following endocrine therapy. After antiestrogen treatment of MCF-7 breast cancer cells, cyclin E2 mRNA and protein were downregulated and cyclin E2-CDK2 activity decreased. However, this regulation was lost in tamoxifen-resistant (MCF-7 TAMR) cells, which overexpressed cyclin E2. Expression of either cyclin E1 or E2 in T-47D breast cancer cells conferred acute antiestrogen resistance, suggesting that cyclin E overexpression contributes to the antiestrogen resistance of tamoxifen-resistant cells. Ectopic expression of cyclin E1 or E2 also reduced sensitivity to CDK4, but not CDK2, inhibition. Proliferation of tamoxifen-resistant cells was inhibited by RNAi-mediated knockdown of cyclin E1, cyclin E2, or CDK2. Furthermore, CDK2 inhibition of E-cyclin overexpressing cells and tamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4 inhibition. Cyclin E2 overexpression is therefore a potential mechanism of resistance to both endocrine therapy and CDK4 inhibition. CDK2 inhibitors hold promise as a component of combination therapies in endocrine-resistant disease as they effectively inhibit cyclin E1 and E2 overexpressing cells and enhance the efficacy of other therapeutics.
Molecular Cancer Therapeutics 05/2012; 11(7):1488-99. DOI:10.1158/1535-7163.MCT-11-0963 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cell cycle is a tightly regulated series of events that governs cell replication and division. Deregulation of cell cycle kinases, e.g., cyclin-dependent kinases (CDKs), can initiate a hyper-proliferative cell phenotype and cause genomic instability, thus facilitating malignant transformation. Pharmacological agents targeting CDKs have been developed as potential anti-cancer agents for over 20 years, evolving from early pan-CDK inhibitors to second-generation inhibitors with much greater specificity and selectivity. Despite these advances in drug design and highly successful preclinical investigations, CDK inhibitors have yet to achieve their expected efficacy in clinical trials. In addition, inhibitors of other cell cycle kinases are currently progressing through clinical trials. Recent biochemical and genetic studies might be used to improve the effectiveness of cell cycle kinase inhibitors as anti-cancer agents through better drug design, therapeutic combinations, and patient selection.
Critical reviews in oncogenesis 01/2012; 17(2):175-98. DOI:10.1615/CritRevOncog.v17.i2.40
[Show abstract][Hide abstract] ABSTRACT: Cyclin D1, and to a lesser extent the other D-type cyclins, is frequently deregulated in cancer and is a biomarker of cancer phenotype and disease progression. The ability of these cyclins to activate the cyclin-dependent kinases (CDKs) CDK4 and CDK6 is the most extensively documented mechanism for their oncogenic actions and provides an attractive therapeutic target. Is this an effective means of targeting the cyclin D oncogenes, and how might the patient subgroups that are most likely to benefit be identified?
Nature Reviews Cancer 07/2011; 11(8):558-72. DOI:10.1038/nrc3090 · 37.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antihormones are of substantial benefit in treating oestrogen receptor-α positive (ER+) breast cancer. However, their anti-tumour
effect is limited by emergence of resistance. Our in vitro studies are highlighting a new underlying concept: that antihormones
are not passive bystanders but alongside growth inhibitory effects promote adverse compensatory mechanisms within tumour cells.
These mechanisms involve drug-induction of signalling elements normally suppressed by oestrogen (E2)-occupied E R˜ While best
exemplified by the tyrosine kinases epidermal growth factor receptor and HER2, microarrays reveal the true diversity of the
induced signalling kinases, where their potential to promote resistance is exacerbated under paracrine conditions. Such drug-induced
events permit initial ER+ breast cancer cell survival, allow development and maintenance of resistance, and also promote gain
of invasiveness under conditions of poor intercellular contact. In addition, prolonged antihormonal exposure is associated
with epigenetic silencing of classical E2-induced tumour suppressors, an event which further contributes to resistance. Based
on proof of principle experiments targeting induced signalling events alongside antihormones or restoring E2-induced suppressor
genes through DNA methylation inhibitor-containing strategies, it is our belief that continued deciphering of these mechanisms
will reveal improved treatments for breast cancer.