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.

Epigenome erosion and SOX10 drive neural crest phenotypic mimicry in triple-negative breast cancer.

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.

The importance of targeting signalling mechanisms of the SLC39A family of zinc transporters to inhibit endocrine resistant breast cancer.

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.

The ZIP6/ZIP10 heteromer is essential for the zinc-mediated trigger of mitosis.

Zinc has been known to be essential for cell division for over 40 years but the molecular pathways involved remain elusive. Cellular zinc import across biological membranes necessitates the help of zinc transporters such as the SLC39A family of ZIP transporters. We have discovered a molecular process that explains why zinc is required for cell division, involving two highly regulated zinc transporters, as a heteromer of ZIP6 and ZIP10, providing the means of cellular zinc entry at a specific time of the cell cycle that initiates a pathway resulting in the onset of mitosis. Crucially, when the zinc influx across this heteromer is blocked by ZIP6 or ZIP10 specific antibodies, there is no evidence of mitosis, confirming the requirement for zinc influx as a trigger of mitosis. The zinc that influxes into cells to trigger mitosis additionally changes the phosphorylation state of STAT3 converting it from a transcription factor to a protein that complexes with this heteromer and pS38Stathmin, the form allowing microtubule rearrangement as required in mitosis. This discovery now explains the specific cellular role of ZIP6 and ZIP10 and how they have special importance in the mitosis process compared to other ZIP transporter family members. This finding offers new therapeutic opportunities for inhibition of cell division in the many proliferative diseases that exist, such as cancer.

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.

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

PURPOSE: The STAKT study examined short-term exposure (4.5 days) to the oral selective pan-AKT inhibitor capivasertib (AZD5363) to determine if this drug can reach its therapeutic target in sufficient concentration to significantly modulate key biomarkers of the AKT pathway and tumor proliferation. PATIENTS AND METHODS: STAKT was a two-stage, double-blind, randomized, placebo-controlled, "window-of-opportunity" study in patients with newly diagnosed ER+ invasive breast cancer. Stage 1 assessed capivasertib 480 mg b.i.d. (recommended monotherapy dose) and placebo, and stage 2 assessed capivasertib 360 and 240 mg b.i.d. Primary endpoints were changes from baseline in AKT pathway markers pPRAS40, pGSK3ß, and proliferation protein Ki67. Pharmacologic and pharmacodynamic properties were analyzed from blood sampling, and tolerability by adverse-event monitoring. RESULTS: After 4.5 days' exposure, capivasertib 480 mg b.i.d. (n = 17) produced significant decreases from baseline versus placebo (n = 11) in pGSK3ß (H-score absolute change: -55.3, P = 0.006) and pPRAS40 (-83.8, P < 0.0001), and a decrease in Ki67 (absolute change in percentage positive nuclei: -9.6%, P = 0.031). Significant changes also occurred in secondary signaling biomarker pS6 (-42.3, P = 0.004), while pAKT (and nuclear FOXO3a) also increased in accordance with capivasertib's mechanism (pAKT: 81.3, P = 0.005). At doses of 360 mg b.i.d. (n = 5) and 240 mg b.i.d. (n = 6), changes in primary and secondary biomarkers were also observed, albeit of smaller magnitude. Biomarker modulation was dose and concentration dependent, and no new safety signals were evident. CONCLUSIONS: Capivasertib 480 mg b.i.d. rapidly modulates key biomarkers of the AKT pathway and decreases proliferation marker Ki67, suggesting future potential as an effective therapy in AKT-dependent breast cancers.

Activated zinc transporter ZIP7 as an indicator of anti-hormone resistance in breast cancer.

ZIP7, a member of the ZIP family of zinc importers, resides on the endoplasmic reticulum membrane and transports zinc from intracellular stores to the cytoplasm after activation by CK2 phosphorylation on two serine residues (S275 and S276). ZIP7 is known to be required for the growth of anti-hormone resistant breast cancer models, especially those with acquired tamoxifen resistance developed from MCF-7. Using our new pS275S276ZIP7 antibody which only recognises activated ZIP7 (pZIP7), we have demonstrated that the hyperactivation of ZIP7 is prevalent in tamoxifen-resistant breast cancer cells. This evidence suggests that pZIP7 might have potential as a biomarker of acquired resistance to such anti-hormones in breast cancer, a current unmet clinical need. In this regard, we have also developed a new immunohistochemical assay for pZIP7 which allowed pZIP7 to be tested on a small clinical series of breast cancer tissues confirming its prevalence in such tumours and relationship to a variety of clinicopathological parameters and biomarkers previously associated with endocrine resistant phenotypes, notably increased activated MAPK signalling, expression of ErbB2, CD71 and the proto-oncogene c-Fos, as well as with increased tumour grade.

MicroRNA-196a is regulated by ER and is a prognostic biomarker in ER+ breast cancer.

BACKGROUND: MicroRNAs are potent post-transcriptional regulators involved in all hallmarks of cancer. Mir-196a is transcribed from two loci and has been implicated in a wide range of developmental and pathogenic processes, with targets including Hox, Fox, Cdk inhibitors and annexins. Genetic variants and altered expression of MIR196A are associated with risk and progression of multiple cancers including breast cancer, however little is known about the regulation of the genes encoding this miRNA, nor the impact of variants therein. METHODS: Genomic data and chromatin interaction analysis were used to discover functional promoter and enhancer elements for MIR196A. Expression data were used to associate MIR196A with mechanisms of resistance, breast cancer subtypes and prognosis. RESULTS: Here we demonstrate that MIR196A displays complex and dynamic expression patterns, in part controlled by long-range transcriptional regulation between promoter and enhancer elements bound by ERα. Expression of this miRNA is significantly increased in drug-resistant models of hormone-receptor positive disease. The expression of MIR196A also proves to be a robust prognostic factor for patients with advanced and post-menopausal ER+ disease. CONCLUSION: This work sheds light on the normal and abnormal regulation of MIR196A and provides a novel stratification method for therapeutically resistant breast cancer.

Correction to: Heregulin ß1 drives gefitinib-resistant growth and invasion in tamoxifen-resistant MCF-7 breast cancer cells.

After the publication of this work [1], an error was noticed in Fig. 2b and Fig. 4b as well as Fig. 4b. and Fig. 5d. Images of the ERK1/2 blots were accidentally duplicated. In Fig. 5a. and Fig. 5c., the last lane for p-ERK1/2 was mistakenly cropped out of the final image. The original blot for Fig. 4b., "total EGFR" (or lane 2) is shown below to avoid any misunderstanding of the data. We apologize for this error, which did not affect any of the interpretations or conclusions of the article.

Long-range regulators of the lncRNA HOTAIR enhance its prognostic potential in breast cancer.

Predicting response to endocrine therapy and survival in oestrogen receptor positive breast cancer is a significant clinical challenge and novel prognostic biomarkers are needed. Long-range regulators of gene expression are emerging as promising biomarkers and therapeutic targets for human diseases, so we have explored the potential of distal enhancer elements of non-coding RNAs in the prognostication of breast cancer survival. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis and is predictive of decreased survival. Here, we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments transcription. This enhancer is dependent on Forkhead-Box transcription factors and functionally interacts with a novel alternate HOTAIR promoter. HOTAIR expression is negatively regulated by oestrogen, positively regulated by FOXA1 and FOXM1, and is inversely correlated with oestrogen receptor and directly correlated with FOXM1 in breast tumours. The combination of HOTAIR and FOXM1 enables greater discrimination of endocrine therapy responders and non-responders in patients with oestrogen receptor positive breast cancer. Consistent with this, HOTAIR expression is increased in cell-line models of endocrine resistance. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in aggressive HER2+ breast tumours. Our study elucidates the transcriptional regulation of HOTAIR, identifies HOTAIR and its regulators as novel biomarkers of patient response to endocrine therapy and corroborates the importance of transcriptional enhancers in cancer.

Biological effects of fulvestrant on estrogen receptor positive human breast cancer: short, medium and long-term effects based on sequential biopsies.

We report the first study of the biological effect of fulvestrant on ER positive clinical breast cancer using sequential biopsies through to progression. Thirty-two locally/systemically advanced breast cancers treated with first-line fulvestrant (250 mg/month) were biopsied at therapy initiation, 6 weeks, 6 months and progression and immunohistochemically-analyzed for Ki67, ER, EGFR and HER2 expression/signaling activity. This series showed good fulvestrant responses (duration of response [DoR] = 25.8 months; clinical benefit = 81%). Ki67 fell (p < 0.001) in 79% of tumours by 6 months and lower Ki67 at all preprogression time-points predicted for longer DoR. ER and PR significantly decreased in all tumours by 6 months (p < 0.001), with some declines in ER (serine 118) phosphorylation and Bcl-2 (p = 0.007). There were modest HER2 increases (p = 0.034, 29% tumours) and loss of any detectable EGFR phosphorylation (p = 0.024, 50% tumours) and MAP kinase (ERK1/2) phosphorylation (p = 0.019, 65% tumours) by 6 months. While ER remained low, there was some recovery of Ki67, Bcl-2 and (weakly) EGFR/MAPK activity in 45-67% patients at progression. Fulvestrant's anti-proliferative impact is related to DoR, but while commonly downregulating ER and indicators of its signaling and depleting EGFR/MAPK signaling in some patients, additional elements must determine response duration. Residual ER at fulvestrant relapse explains reported sensitivity to further endocrine therapies. Occasional modest treatment-induced HER2 and weakly detectable EGFR/HER2/MAPK signaling at relapse suggests targeting of such activity might have value alongside fulvestrant in some patients. However, unknown pathways must drive relapse in most. Ki67 has biomarker potential to predict fulvestrant outcome and as a quantitative measure of response.

ELF5 Drives Lung Metastasis in Luminal Breast Cancer through Recruitment of Gr1+ CD11b+ Myeloid-Derived Suppressor Cells.

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.

DNA methylation of oestrogen-regulated enhancers defines endocrine sensitivity in breast cancer.

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.

A good drug made better: the fulvestrant dose-response story.

Sequential use of endocrine therapies remains the cornerstone of treatment for hormone receptor-positive advanced breast cancer, before the use of cytotoxic chemotherapy for unresponsive disease. Fulvestrant is an estrogen receptor (ER) antagonist approved for the treatment of postmenopausal women with ER+ advanced breast cancer after failure of prior antiestrogen therapy. Initially approved at a monthly dose of 250 mg, the recommended fulvestrant dose was revised to 500 mg (500 mg/mo plus 500 mg on day 14 of month 1) after demonstration of improved progression-free survival versus fulvestrant 250 mg. We have reviewed the dose-dependent effects of fulvestrant, both from a retrospective combined analysis of dose-dependent reduction of tumor biomarkers in the presurgical setting (3 previously reported studies: Study 18, Neoadjuvant Endocrine Therapy for Women with Estrogen-Sensitive Tumors, and Trial 57) and from a review of clinical studies for advanced breast cancer in postmenopausal women. Analysis of presurgical data revealed a consistent dose-dependent effect for fulvestrant on tumor biomarkers, with increasing fulvestrant dose resulting in greater reductions in ER, progesterone receptor, and Ki67 labeling index. The dose-dependent biological effect corresponds with the dose-dependent clinical efficacy observed in the treatment of advanced breast cancer after failure of prior antiestrogen therapy. Although it remains to be determined in a phase III trial, cross-trial comparisons suggest a dose-dependent relationship for fulvestrant as first-line treatment for advanced breast cancer. Overall, biological and clinical data demonstrate a strong dose-dependent relationship for fulvestrant, supporting the efficacy benefit seen with fulvestrant 500 mg over the 250 mg dose.

Impact of dual mTORC1/2 mTOR kinase inhibitor AZD8055 on acquired endocrine resistance in breast cancer in vitro.

INTRODUCTION: Upregulation of PI3K/Akt/mTOR signalling in endocrine-resistant breast cancer (BC) has identified mTOR as an attractive target alongside anti-hormones to control resistance. RAD001 (everolimus/Afinitor®), an allosteric mTOR inhibitor, is proving valuable in this setting; however, some patients are inherently refractory or relapse during treatment requiring alternative strategies. Here we evaluate the potential for novel dual mTORC1/2 mTOR kinase inhibitors, exemplified by AZD8055, by comparison with RAD001 in ER + endocrine resistant BC cells. METHODS: In vitro models of tamoxifen (TamR) or oestrogen deprivation resistance (MCF7-X) were treated with RAD001 or AZD8055 alone or combined with anti-hormone fulvestrant. Endpoints included growth, cell proliferation (Ki67), viability and migration, with PI3K/AKT/mTOR signalling impact monitored by Western blotting. Potential ER cross-talk was investigated by immunocytochemistry and RT-PCR. RESULTS: RAD001 was a poor growth inhibitor of MCF7-derived TamR and MCF7-X cells (IC50 ≥1 µM), rapidly inhibiting mTORC1 but not mTORC2/AKT signalling. In contrast AZD8055, which rapidly inhibited both mTORC1 and mTORC2/AKT activity, was a highly effective (P <0.001) growth inhibitor of TamR (IC50 18 nM) and MCF7-X (IC50 24 nM), and of a further T47D-derived tamoxifen resistant model T47D-tamR (IC50 19 nM). AZD8055 significantly (P <0.05) inhibited resistant cell proliferation, increased cell death and reduced migration. Furthermore, dual treatment of TamR or MCF7-X cells with AZD8055 plus fulvestrant provided superior control of resistant growth versus either agent alone (P <0.05). Co-treating with AZD8055 alongside tamoxifen (P <0.01) or oestrogen deprivation (P <0.05) also effectively inhibited endocrine responsive MCF-7 cells. Although AZD8055 inhibited oestrogen receptor (ER) ser167 phosphorylation in TamR and MCF7-X, it had no effect on ER ser118 activity or expression of several ER-regulated genes, suggesting the mTOR kinase inhibitor impact was largely ER-independent. The capacity of AZD8055 for ER-independent activity was further evidenced by growth inhibition (IC5018 and 20 nM) of two acquired fulvestrant resistant models lacking ER. CONCLUSIONS: This is the first report demonstrating dual mTORC1/2 mTOR kinase inhibitors have potential to control acquired endocrine resistant BC, even under conditions where everolimus fails. Such inhibitors may prove of particular benefit when used alongside anti-hormonal treatment as second-line therapy in endocrine resistant disease, and also potentially alongside anti-hormones during the earlier endocrine responsive phase to hinder development of resistance.

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer.

INTRODUCTION: Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS: More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS: The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS: With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

BCL-2 hypermethylation is a potential biomarker of sensitivity to antimitotic chemotherapy in endocrine-resistant breast cancer.

Overexpression of the antiapoptotic 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 favorable prognosis, estrogen receptor (ER) positivity, and low tumor grade, whereas 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 tumors, provided a stronger predictor of patient survival (P = 0.019) when compared with 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 antimitotic 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 nonresectable, metastatic disease.

Global characterization of signalling networks associated with tamoxifen resistance in breast cancer.

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 myristoylated alanine-rich C-kinase substrate (MARCKS) were increased two- and eightfold in TamR cells respectively, and these proteins 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 in stratification of patients for optimal therapy.

A randomized trial to assess the biological activity of short-term (pre-surgical) fulvestrant 500 mg plus anastrozole versus fulvestrant 500 mg alone or anastrozole alone on primary breast cancer.

INTRODUCTION: Fulvestrant shows dose-dependent biological activity. Greater estrogen-receptor (ER) blockade may feasibly be achieved by combining fulvestrant with anastrozole. This pre-surgical study compared fulvestrant plus anastrozole versus either agent alone in patients with ER-positive breast cancer. METHODS: In this double-blind, multicenter trial, 121 patients received fulvestrant 500 mg on Day 1 plus anastrozole 1 mg/day for 14 to 21 days (F + A); fulvestrant plus anastrozole placebo (F); or fulvestrant placebo plus anastrozole (A), 2 to 3 weeks before surgery. ER, progesterone-receptor (PgR) and Ki67 expression were determined from tumor biopsies before treatment and at surgery. RESULTS: A total of 103 paired samples were available (F, n = 35; F+A, n = 31; A, n = 37). All treatments significantly reduced mean ER expression from baseline (F: -41%, P = 0.0001; F + A: -39%, P = 0.0001; A: -13%, P = 0.0034). F and F + A led to greater reductions in ER versus A (both P = 0.0001); F + A did not lead to additional reductions versus F. PgR and Ki67 expression were significantly reduced with all treatments (means were -34% to -45%, and -75% to -85%, respectively; all P = 0.0001), with no differences between groups. CONCLUSIONS: In this short-term study, all treatments reduced ER expression, although F and F + A showed greater reductions than A. No significant differences were detected between the treatment groups in terms of PgR and Ki67 expression. No additional reduction in tumor biomarkers with combination treatment was observed, suggesting that F + A is unlikely to have further clinical benefit over F alone. TRIAL REGISTRATION: Clinicaltrials.gov NCT00259090.