Explore the vast range of titles available.

Key Points Endocrine therapies that target oestrogen action (anti-oestrogens and aromatase inhibitors) are widely used and successful breast cancer therapies, but many women treated with these therapies will relapse with endocrine-resistant disease. Mechanisms of endocrine resistance in oestrogen receptor (ER)-positive breast cancers include loss of ERα expression and expression of truncated isoforms of ERα and ERβ, post-translational modifications of ERα, increased AP1 activity and deregulation of ER co-activators, increased receptor tyrosine kinase signalling leading to the activation of the Erk and PI3K pathways, and deregulation of the cell cycle and apoptotic machinery. Gene expression signatures that are predictive of poor outcome in women treated with tamoxifen commonly contain ER target genes, as well as genes involved in proliferation, apoptosis, and invasion and metastasis. Many of these signatures are also predictive of outcome in women who have not been treated with tamoxifen and so are markers of intrinsic biology rather than specific to tamoxifen responsiveness. Gene expression signatures representing particular biological processes (for example, cell cycle progression, cell death and invasion) or pathways (for example, RB deregulation, MYC overexpression and E2f activation) can also predict outcome in women treated with tamoxifen and point towards possible mechanisms for endocrine resistance. Functional genetic screens have successfully identified several genes, the loss or overexpression of which can reduce anti-oestrogen sensitivity in cell lines and is associated with clinical endocrine resistance. Insights into the mechanisms of resistance have suggested possible therapeutic approaches for endocrine-resistant ER-positive breast cancer, for example tyrosine kinase inhibitors. Further potential therapeutic targets may emerge from combining large-scale genomic and transcriptomic data with large-scale functional analyses. The efficacy of endocrine therapies (such as tamoxifen) in breast cancer is limited by intrinsic and acquired therapeutic resistance. What do we know about the genetic lesions and molecular processes that determine endocrine resistance in the clinic, and how can we use this to improve therapy? Endocrine therapies targeting oestrogen action (anti-oestrogens, such as tamoxifen, and aromatase inhibitors) decrease mortality from breast cancer, but their efficacy is limited by intrinsic and acquired therapeutic resistance. Candidate molecular biomarkers and gene expression signatures of tamoxifen response emphasize the importance of deregulation of proliferation and survival signalling in endocrine resistance. However, definition of the specific genetic lesions and molecular processes that determine clinical endocrine resistance is incomplete. The development of large-scale computational and genetic approaches offers the promise of identifying the mediators of endocrine resistance that may be exploited as potential therapeutic targets and biomarkers of response in the clinic.

Key Points Cyclin D–cyclin-dependent kinase 4 (CDK4) or CDK6 activation promotes cell cycle progression through the phosphorylation of substrates, including RB and transcription factors with roles in proliferation and differentiation. These kinase complexes also target substrates with roles in centrosome duplication, mitochondrial function, cell growth, cell adhesion and motility, and cytoskeletal modelling. D-type cyclins have non-catalytic roles in which interactions with chromatin-modifying enzymes and diverse transcription factors, including steroid hormone receptors, leads to the transcriptional regulation of suites of genes that are involved in proliferation and differentiation. Independently of CDK activation, the D-type cyclins also facilitate efficient DNA repair and indirectly activate CDK2 through the sequestration of CDK inhibitors. CCND1 is an established human oncogene that is commonly overexpressed through copy number alterations, or more rarely by mutation, or as a consequence of the deregulation of mitogenic signalling downstream of oncogenes such as ERBB2. CCND1 overexpression causes a number of potentially oncogenic responses in experimental models and is associated with poor patient outcome. Cyclin D1 and its associated CDKs are potential therapeutic targets. Promising results from early CDK inhibitors in experimental systems were not followed by evidence for efficacy in clinical trials. Possible reasons for this disappointing outcome include poor pharmacokinetics, suboptimal dosing schedules and clinical testing in unselected patient populations. Second-generation, more selective inhibitors of CDK4 and CDK6 are now undergoing clinical testing. Possible alternative approaches to targeting cyclin D1 include the use of compounds that affect CCND1 transcription or cyclin D1 protein turnover, and the use of combination therapies that simultaneously target multiple end points of cyclin D1 action. Central to the effective use of these novel approaches is the better selection of patient subgroups that are likely to respond. Is the ability of D-type cyclins to activate cyclin-dependent kinases an effective means of targeting these oncogenes, and how might the patient subgroups that are most likely to benefit be identified? 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?

The highly conserved E-type cyclins are core components of the cell cycle machinery, facilitating the transition into S phase through activation of the cyclin dependent kinases, and assembly of pre-replication complexes on DNA. Cyclin E1 and cyclin E2 are assumed to be functionally redundant, as cyclin E1 -/- E2 -/- mice are embryonic lethal while cyclin E1 -/- and E2 -/- single knockout mice have primarily normal phenotypes. However more detailed studies of the functions and regulation of the E-cyclins have unveiled potential additional roles for these proteins, such as in endoreplication and meiosis, which are more closely associated with either cyclin E1 or cyclin E2. Moreover, expression of each E-cyclin can be independently regulated by distinct transcription factors and microRNAs, allowing for context-specific expression. Furthermore, cyclins E1 and E2 are frequently expressed independently of one another in human cancer, with unique associations to signatures of poor prognosis. These data imply an absence of co-regulation of cyclins E1 and E2 during tumorigenesis and possibly different contributions to cancer progression. This is supported by in vitro data identifying divergent regulation of the two genes, as well as potentially different roles in vivo .

Chronic pancreatitis and pancreatic cancer are characterised by extensive stellate cell mediated fibrosis, and current therapeutic development includes targeting pancreatic cancer stroma and tumor-host interactions. Recent evidence has suggested that circulating bone marrow derived stem cells (BMDC) contribute to solid organs. We aimed to define the role of circulating haematopoietic cells in the normal and diseased pancreas. Whole bone marrow was harvested from male β-actin-EGFP donor mice and transplanted into irradiated female recipient C57/BL6 mice. Chronic pancreatitis was induced with repeat injections of caerulein, while carcinogenesis was induced with an intrapancreatic injection of dimethylbenzanthracene (DMBA). Phenotype of engrafted donor-derived cells within the pancreas was assessed by immunohistochemistry, immunofluorescence and in situ hybridisation. GFP positive cells were visible in the exocrine pancreatic epithelia from 3 months post transplantation. These exhibited acinar morphology and were positive for amylase and peanut agglutinin. Mice administered caerulein developed chronic pancreatitis while DMBA mice exhibited precursor lesions and pancreatic cancer. No acinar cells were identified to be donor-derived upon cessation of cerulein treatment, however rare occurrences of bone marrow-derived acinar cells were observed during pancreatic regeneration. Increased recruitment of BMDC was observed within the desmoplastic stroma, contributing to the activated pancreatic stellate cell (PaSC) population in both diseases. Expression of stellate cell markers CELSR3, PBX1 and GFAP was observed in BMD cancer-associated PaSCs, however cancer-associated, but not pancreatitis-associated BMD PaSCs, expressed the cancer PaSC specific marker CELSR3. This study demonstrates that BMDC can incorporate into the pancreas and adopt the differentiated state of the exocrine compartment. BMDC that contribute to the activated PaSC population in chronic pancreatitis and pancreatic cancer have different phenotypes, and may play important roles in these diseases. Further, bone marrow transplantation may provide a useful model for the study of tumor-host interactions in cancer and pancreatitis.

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.

Breast cancers lacking the estrogen receptor (ER) can be distinguished from other breast cancers on the basis of poor prognosis, high grade, distinctive histopathology and unique molecular signatures. These features further distinguish estrogen receptor negative (ER-) tumor subtypes, but targeted therapy is currently limited to tumors over-expressing the ErbB2 receptor. To uncover the pathways against which future therapies could be developed we undertook a meta-analysis of gene expression from five large microarray datasets relative to ER status. A measure of association with ER status was calculated for every Affymetrix HG-U133A probe set and the pathways that distinguished ER- tumors were defined by testing for enrichment of biologically defined gene sets using Gene Set Enrichment Analysis (GSEA). As expected, the expression of the direct transcriptional targets of the ER was muted in ER- tumors, but the expression of genes indirectly regulated by estrogen was enhanced. We also observed enrichment of independent MYC- and E2F-driven transcriptional programs. We used a cell model of estrogen and MYC action to define the interaction between estrogen and MYC transcriptional activity in breast cancer. We found that the basal subgroup of ER- breast cancer showed a strong MYC transcriptional response that reproduced the indirect estrogen response seen in estrogen receptor positive (ER+) breast cancer cells. Increased transcriptional activity of MYC is a characteristic of basal breast cancers where it mimics a large part of an estrogen response in the absence of the ER, suggesting a mechanism by which these cancers achieve estrogen-independence and providing a potential therapeutic target for this poor prognosis sub group of breast cancer.

Estrogen is a pivotal regulator of cell proliferation in the normal breast and breast cancer. Endocrine therapies targeting the estrogen receptor are effective in breast cancer, but their success is limited by intrinsic and acquired resistance. With the goal of gaining mechanistic insights into estrogen action and endocrine resistance, we classified estrogen-regulated genes by function, and determined the relationship between functionally-related genesets and the response to tamoxifen in breast cancer patients. Estrogen-responsive genes were identified by transcript profiling of MCF-7 breast cancer cells. Pathway analysis based on functional annotation of these estrogen-regulated genes identified gene signatures with known or predicted roles in cell cycle control, cell growth (i.e. ribosome biogenesis and protein synthesis), cell death/survival signaling and transcriptional regulation. Since inducible expression of c-Myc in antiestrogen-arrested cells can recapitulate many of the effects of estrogen on molecular endpoints related to cell cycle progression, the estrogen-regulated genes that were also targets of c-Myc were identified using cells inducibly expressing c-Myc. Selected genes classified as estrogen and c-Myc targets displayed similar levels of regulation by estrogen and c-Myc and were not estrogen-regulated in the presence of siMyc. Genes regulated by c-Myc accounted for 50% of all acutely estrogen-regulated genes but comprised 85% (110/129 genes) in the cell growth signature. siRNA-mediated inhibition of c-Myc induction impaired estrogen regulation of ribosome biogenesis and protein synthesis, consistent with the prediction that estrogen regulates cell growth principally via c-Myc. The 'cell cycle', 'cell growth' and 'cell death' gene signatures each identified patients with an attenuated response in a cohort of 246 tamoxifen-treated patients. In multivariate analysis the cell death signature was predictive independent of the cell cycle and cell growth signatures. These functionally-based gene signatures can stratify patients treated with tamoxifen into groups with differing outcome, and potentially identify distinct mechanisms of tamoxifen resistance.

Chromosome locus 11q13 is frequently amplified in a number of human cancers including carcinoma of the breast where up to 15% carry this chromosomal abnormality. Originally 11q13 amplification was thought to involve a single amplicon spanning many megabases, but more recent data have identified four core regions within 11q13 that can be amplified independently or together in different combinations. Although the region harbors several genes with known or suspected oncogenic potential, the complex structure of the amplicons and the fact that 11q13 is gene-rich have made definitive identification of specific genes that contribute to the genesis and progression of breast cancer a difficult and continuing process. To date CCND1, encoding the cell cycle regulatory gene cyclin D1, and EMS1, encoding the filamentous actin binding protein and c-Src substrate cortactin, are the favored candidates responsible for the emergence of two of the four amplification cores.

Background Although MYC is an attractive therapeutic target for breast cancer treatment, it has proven challenging to inhibit MYC directly, and clinically effective pharmaceutical agents targeting MYC are not yet available. An alternative approach is to identify genes that are synthetically lethal in MYC-dependent cancer. Recent studies have identified several cell cycle kinases as MYC synthetic-lethal genes. We therefore investigated the therapeutic potential of specific cyclin-dependent kinase (CDK) inhibition in MYC-driven breast cancer. Methods Using small interfering RNA (siRNA), MYC expression was depleted in 26 human breast cancer cell lines and cell proliferation evaluated by BrdU incorporation. MYC-dependent and MYC-independent cell lines were classified based on their sensitivity to siRNA-mediated MYC knockdown. We then inhibited CDKs including CDK4/6, CDK2 and CDK1 individually using either RNAi or small molecule inhibitors, and compared sensitivity to CDK inhibition with MYC dependence in breast cancer cells. Results Breast cancer cells displayed a wide range of sensitivity to siRNA-mediated MYC knockdown. The sensitivity was correlated with MYC protein expression and MYC phosphorylation level. Sensitivity to siRNA-mediated MYC knockdown did not parallel sensitivity to the CDK4/6 inhibitor PD0332991; instead MYC-independent cell lines were generally sensitive to PD0332991. Cell cycle arrest induced by MYC knockdown was accompanied by a decrease in CDK2 activity, but inactivation of CDK2 did not selectively affect the viability of MYC-dependent breast cancer cells. In contrast, CDK1 inactivation significantly induced apoptosis and reduced viability of MYC-dependent cells but not MYC- independent cells. This selective induction of apoptosis by CDK1 inhibitors was associated with up-regulation of the pro-apoptotic molecule BIM and was p53-independent. Conclusions Overall, these results suggest that further investigation of CDK1 inhibition as a potential therapy for MYC-dependent breast cancer is warranted.

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. The molecular factors influencing patient response to endocrine therapy are poorly understood. Here Stone et al. characterize the DNA methylome of endocrine response and show that methylation of oestrogen receptor-associated enhancers underpins endocrine sensitivity in human breast cancer.