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.

Figure S1: Endocrine-resistant MCF-7 cells demonstrate sensitivity to TRAIL Figure S2: Endocrine-resistant CSCs demonstrate sensitivity to TRIAL. Figure S3: Primary-derived patient samples. Figure S4: Endocrine-resistant primary and PDX tumour cells are sensitive to TRAIL. Figure S5: Mechanism of endocrine-resistant tumour cell sensitivity to TRAIL.

Aromatase (CYP19A1) inhibitors are the mainstay therapeutics for the treatment of hormone dependant breast cancer, which accounts for approximately 70% of all breast cancer cases. However, increased resistance to the clinically used aromatase inhibitors, including letrozole and anastrazole, and off target effects, necessitates the development of aromatase inhibitors with improved drug profiles. The development of extended 4th generation pyridine based aromatase inhibitors with dual binding (haem and access channel) is therefore of interest and here we describe the design, synthesis and computational studies. Cytotoxicity and selectivity studies identified the pyridine derivative (4-bromophenyl)(6-(but-2-yn-1-yloxy)benzofuran-2-yl)(pyridin-3-yl)methanol (10c) as optimal with CYP19A1 IC50 0.83 nM (c.f. letrozole IC50 0.70 nM), and an excellent cytotoxicity and selectivity profile. Interestingly, computational studies for the 6-O-butynyloxy (10) and 6-O-pentynyloxy (11) derivatives identified an alternative access channel lined by Phe221, Trp224, Gln225 and Leu477, providing further insight into the potential binding mode and interactions of the non-steroidal aromatase inhibitors.

PDF file, 78KB, Supplementary Figure 2: Deregulation of cyclin E in tamoxifen-resistant cells. MCF-7C and TAMR cells were treated with vehicle, OH-Tam (100 nM) or fulvestrant (10 nM). A: S phase percentage derived from flow cytometry of propidium iodide-stained cells. B: Densitometry of Western blots using GAPDH as a loading control. Data represent the mean range of duplicate experiments.

Background FURVA, a randomised, double-blind Phase II trial, investigated whether the addition of vandetanib to fulvestrant improved progression-free survival (PFS) in patients with an aromatase inhibitor(AI)-resistant advanced breast cancer. Methods Postmenopausal women with oestrogen receptor-positive (ER+ve)/HER2-negative advanced breast cancer, who experienced disease progression on an AI, were randomised (1:1) to fulvestrant 500 mg (Q28) with vandetanib 300 mg od (f + v) or placebo (f + p) until disease progression or discontinuation. The primary endpoint was PFS; secondary endpoints included overall survival (OS) and the influence of REarranged during Transfection (RET) signalling on outcomes. Results In total, 165 participants were randomised to f + v ( n = 80) or f + p ( n = 85). Median PFS was 5.5 months (m) for f + v compared to 5.5 m for f + p (hazard ratio (HR) 0.88; 95% CI: 0.62–1.23; P = 0.22). Unexpectedly, high total RET expression was associated with a PFS advantage of 8.87 m vs 3.94 with low RET (HR 0.493: 95% CI 0.32–0.77; P = 0.002) independent of the treatment arm, supported by an OS advantage 21.95 m vs 18.04 (HR 0.584; 95% CI 0.34–1.00; P = 0.051) in the high-RET group. Conclusion The addition of vandetanib to fulvestrant does not improve PFS. However, high total RET expression was associated with improved PFS, suggesting RET may have a prognostic role in patients treated with fulvestrant. Clinical trial registration ClinicalTrials.gov, NCT02530411.

PDF - 4365KB, BCL-2 Methylation vs disease free survival.

PDF - 80KB, The effect of BI2536 in endocrine-sensitive and endocrine-resistant breast cancer cells.

PDF - 289KB, BCL-2 450K Methylation array probes (n=42).

PDF - 664KB, BCL-2 Gene expression vs methylation status.

PDF - 221KB, A. Plk1 Expression vs relapse-free survival in endocrine treated ER-positive breast cancer patients (n=287). B. Correlative analysis of ER, BCL-2 and PLK1 gene expression in TCGA breast cohort (n=774).

Supplementary Data from Proliferation and AKT Activity Biomarker Analyses after Capivasertib (AZD5363) Treatment of Patients with ER+ Invasive Breast Cancer (STAKT)

Age is the biggest risk factor for Alzheimer’s disease (AD) but being female also increases risk with ∼63% of AD patients being female. This sex difference could partly be explained by menopause-related oestrogen deficiencies during ageing. Oestrogen is neuroprotective and it is highly probable this deficiency after the menopause affects neuronal survival and contributes to the onset of AD. Oestrogen receptors are found in mitochondria and evidence suggests that the neuroprotective effect of oestrogen involves supporting mitochondrial function. The clear link between mitochondrial dysfunction and the pathophysiology of AD suggests a decrease in the beneficial effects of oestrogen on mitochondrial function after the menopause would have serious consequences. We hypothesise that alterations in oestrogen, due to ageing and the menopause, affect mitochondrial function and contribute to the increased susceptibility of women to AD. Levels of mitochondrial proteins, dynamin-related protein-1 (DRP-1), mitofusin-1/2 (Mfn1/2), mitochondrial dynamin-like GTPase (OPA1) and the electron transport chain (ETC) complexes 1-5, were measured using immunoblotting in frontal cortical brain samples from young (20-36), middle-aged (40-53) and old (64-99) men and women with no history of dementia and in AD patients (Braak stage 5/6, 69-92 age). In female AD brains a significant decrease was observed in Mnf1 and DRP-1 compared to age-matched females and in OPA1 in male AD compared to old male brains. Mnf2 and ETC complex 3 (cytochrome reductase) were significantly increased in female AD compared to male AD brains. DRP-1 was significantly decreased in old males compared to middle-aged males, while ETC complex 2 (succinate dehydrogenase) was significantly increased in old females compared to young females. We demonstrate for the first time age, sex and disease differences in the expression of proteins involved in various aspects of mitochondrial function including fusion, fission and energy production. These changes could underlie previous findings showing a range of mitochondrial abnormalities in AD brains and support the ‘mitochondrial cascade hypothesis’ that mitochondrial dysfunction is the primary event causing β-amyloid deposition, synaptic degeneration, and NFT formation (Swerdlow et al., J. Alz. Dis. 2010 20:Suppl 2, 265-79.). Our findings could partly explain female vulnerability to AD development.

PDF file, 41KB, Supplementary Figure 3: Effect of overexpression of cyclins E1 and E2 on antiestrogen-induced growth arrest. T-47D cells overexpressing cyclin E1, cyclin E2 or vector control were treated with fulvestrant or vehicle. Densitometry of Western blots of cell lysates collected after 24h (A) or 48h (B) fulvestrant treatment. Loading was corrected using β-actin levels. Data represent mean SEM of quadruplicate experiments.

PDF - 459KB, BCL-2 Methylation status in cancer vs normal tissue.

PDF - 51KB, PLK1 gene expression/survival analysis.

PDF file, 929KB, Supplementary Figure 1: Cyclin E2 association with breast cancer subtype, grade, and outcome. A: Relationship between a second cyclin E2 probeset and breast cancer subtype, or histological grade. Box: upper and lower quartiles; dividing line: median. B: Relative expression of cyclin E1 and a second cyclin E2 probeset in individual tumours in breast cancer subtypes. C: Kaplan-Meier survival analysis of distant metastasis-free survival from ER-positive patients treated with endocrine therapy (n=287) and untreated ER-negative patients (n=106), using a 70% cutoff between high and low expression.

Aim: Zinc is a key secondary messenger that can regulate multiple signalling pathways within cancer cells, thus its levels need to be strictly controlled. The Zrt, Irt-like protein (ZIP, SLC39A) family of zinc transporters increase cytosolic zinc from either extracellular or intracellular stores. This study examines the relevance of zinc transporters ZIP7 and ZIP6 as therapeutic targets in tamoxifen resistant (TAMR) breast cancer. Methods: A series of in vitro assays, including immunohistochemistry, immunofluorescence, flow cytometry, and western blotting were used to evaluate levels and activity of ZIP7 and ZIP6 in models of TAMR and sensitive (MCF-7) breast cancer. Analyses of these transporters in the clinical setting were performed using publicly available online resources: Gene Expression Profiling Interactive Analysis (GEPIA)2 and Kaplan-Meier Plotter (KmPlot). Results: Both total and activated levels of ZIP7 were significantly elevated in TAMR cells versus responsive MCF-7 cells. This was accompanied by an associated increase in free cytoplasmic zinc leading to amplification of downstream signals. Consistent with our proposed model, activated ZIP6 levels correlated with mitotic cells, which could be efficiently inhibited through use of our anti-ZIP6 monoclonal antibody. Mitotic inhibition translated to impaired proliferation in both models, with TAMR cells displaying increased sensitivity. Analysis of matched tumour and normal breast samples from patients revealed significant increases in both ZIP7 and ZIP6 in tumours, as well as family member ZIP4. Kaplan-Meier analysis revealed that high ZIP7 levels correlated with decreased overall and relapse-free survival (RFS) of patients, including patient groups who had received systemic endocrine therapy or tamoxifen only. In contrast, high ZIP6 levels were significantly linked to improved overall and RFS in all patients, as well as RFS in patients that received systemic endocrine therapy. Conclusions: TAMR cells displayed increased activity of both ZIP7 and ZIP6 transporters compared to anti-hormone responsive cells, suggesting their potential as novel therapeutic targets following development of resistant disease.

Intratumoral heterogeneity is caused by genomic instability and phenotypic plasticity, but how these features co-evolve remains unclear. SOX10 is a neural crest stem cell (NCSC) specifier and candidate mediator of phenotypic plasticity in cancer. We investigated its relevance in breast cancer by immunophenotyping 21 normal breast and 1860 tumour samples. Nuclear SOX10 was detected in normal mammary luminal progenitor cells, the histogenic origin of most TNBCs. In tumours, nuclear SOX10 was almost exclusive to TNBC, and predicted poorer outcome amongst cross-sectional (p = 0.0015, hazard ratio 2.02, n = 224) and metaplastic (p = 0.04, n = 66) cases. To understand SOX10's influence over the transcriptome during the transition from normal to malignant states, we performed a systems-level analysis of co-expression data, de-noising the networks with an eigen-decomposition method. This identified a core module in SOX10's normal mammary epithelial network that becomes rewired to NCSC genes in TNBC. Crucially, this reprogramming was proportional to genome-wide promoter methylation loss, particularly at lineage-specifying CpG-island shores. We propose that the progressive, genome-wide methylation loss in TNBC simulates more primitive epigenome architecture, making cells vulnerable to SOX10-driven reprogramming. This study demonstrates potential utility for SOX10 as a prognostic biomarker in TNBC and provides new insights about developmental phenotypic mimicry-a major contributor to intratumoral heterogeneity.

One in every eight women will be diagnosed with breast cancer during their lifetime and approximately 70% of all patients are oestrogen receptor (ER) positive depending upon oestrogen for their growth accounting for third generation aromatase (CYP19A1) inhibitors being the mainstay in the treatment of ER-positive breast cancer. Despite the success of current aromatase inhibitors, acquired resistance occurs after prolonged therapy. Although the precise mechanisms of resistance are not known, lack of cross resistance among aromatase inhibitors drives the need for a newer generation of inhibitors to overcome this resistance alongside minimising toxicity and adverse effects. Novel triazole-based inhibitors were designed based on previously published parent compound 5a, making use of the now available crystal structure of CYP19A1 (PDB 3S79), to make modifications at specific sites to explore the potential of dual binding at both the active site and the access channel. Modifications included adding long chain substituents e.g. but-2-ynyloxy and pent-2-ynyloxy at different positions including the most active compound 13h with IC50 value in the low picomolar range (0.09 nM). Aromatase inhibition results paired with molecular dynamics studies provided a clear structure activity relationship and favourable dual binding mode was verified. Toxicity assays and CYP selectivity profile studies for some example compounds were performed to assess the safety profile of the prepared inhibitors providing the basis for the 4th generation nonsteroidal aromatase inhibitors.