TRPS1 regulates oestrogen receptor binding and histone acetylation at enhancers.

The chromatin state is finely tuned to regulate function and specificity for transcription factors such as oestrogen receptor alpha (ER), which contributes to cell growth in breast cancer. ER transcriptional potential is mediated, in large part, by the specific associated proteins and co-factors that interact with it. Despite the identification and characterisation of several ER coregulators, a complete and systematic view of ER-regulating chromatin modifiers is lacking. By exploiting a focused siRNA screen that investigated the requirement for a library of 330 chromatin regulators in ER-mediated cell growth, we find that the NuRD and coREST histone deacetylation complexes are critical for breast cancer cell proliferation. Further, by proteomic and genomics approaches, we discover the transcription factor TRPS1 to be a key interactor of the NuRD and coREST complexes. Interestingly, TRPS1 gene amplification occurs in 28% of human breast tumours and is associated with poor prognosis. We propose that TRPS1 is required to repress spurious binding of ER, where it contributes to the removal of histone acetylation. Our data suggest that TRPS1 is an important ER-associated transcriptional repressor that regulates cell proliferation, chromatin acetylation and ER binding at the chromatin of cis-regulatory elements.

Cell cycle-coupled expansion of AR activity promotes cancer progression.

The androgen receptor (AR) is required for prostate cancer (PCa) survival and progression, and ablation of AR activity is the first line of therapeutic intervention for disseminated disease. While initially effective, recurrent tumors ultimately arise for which there is no durable cure. Despite the dependence of PCa on AR activity throughout the course of disease, delineation of the AR-dependent transcriptional network that governs disease progression remains elusive, and the function of AR in mitotically active cells is not well understood. Analyzing AR activity as a function of cell cycle revealed an unexpected and highly expanded repertoire of AR-regulated gene networks in actively cycling cells. New AR functions segregated into two major clusters: those that are specific to cycling cells and retained throughout the mitotic cell cycle ('Cell Cycle Common'), versus those that were specifically enriched in a subset of cell cycle phases ('Phase Restricted'). Further analyses identified previously unrecognized AR functions in major pathways associated with clinical PCa progression. Illustrating the impact of these unmasked AR-driven pathways, dihydroceramide desaturase 1 was identified as an AR-regulated gene in mitotically active cells that promoted pro-metastatic phenotypes, and in advanced PCa proved to be highly associated with development of metastases, recurrence after therapeutic intervention and reduced overall survival. Taken together, these findings delineate AR function in mitotically active tumor cells, thus providing critical insight into the molecular basis by which AR promotes development of lethal PCa and nominate new avenues for therapeutic intervention.

Mechanisms of oestrogen receptor (ER) gene regulation in breast cancer.

Most breast cancers are driven by a transcription factor called oestrogen receptor (ER). Understanding the mechanisms of ER activity in breast cancer has been a major research interest and recent genomic advances have revealed extraordinary insights into how ER mediates gene transcription and what occurs during endocrine resistance. This review discusses our current understanding on ER activity, with an emphasis on several evolving, but important areas of ER biology.

Genomic interaction between ER and HMGB2 identifies DDX18 as a novel driver of endocrine resistance in breast cancer cells.

Breast cancer resistance to endocrine therapies such as tamoxifen and aromatase inhibitors is a significant clinical problem. Steroid receptor coactivator-1 (SRC-1), a coregulatory protein of the oestrogen receptor (ER), has previously been shown to have a significant role in the progression of breast cancer. The chromatin protein high mobility group box 2 (HMGB2) was identified as an SRC-1 interacting protein in the endocrine-resistant setting. We investigated the expression of HMGB2 in a cohort of 1068 breast cancer patients and found an association with increased disease-free survival time in patients treated with endocrine therapy. However, it was also verified that HMGB2 expression could be switched on in endocrine-resistant tumours from breast cancer patients. To explore the function of this poorly characterized protein, we performed HMGB2 ChIPseq and found distinct binding patterns between the two contexts. In the resistant setting, the HMGB2, SRC-1 and ER complex are enriched at promoter regions of target genes, with bioinformatic analysis indicating a switch in binding partners between the sensitive and resistant phenotypes. Integration of binding and gene expression data reveals a concise set of target genes of this complex including the RNA helicase DDX18. Modulation of DDX18 directly affects growth of tamoxifen-resistant cells, suggesting that it may be a critical downstream effector of the HMGB2:ER complex. This study defines HMGB2 interactions with the ER complex at specific target genes in the tamoxifen-resistant setting.

Elevated levels of FOXA1 facilitate androgen receptor chromatin binding resulting in a CRPC-like phenotype.

Castration-resistant prostate cancer (CRPC) continues to pose a significant clinical challenge with new generation second-line hormonal therapies affording limited improvement in disease outcome. As the androgen receptor (AR) remains a critical driver in CRPC, understanding the determinants of its transcriptional activity is important for developing new AR-targeted therapies. FOXA1 is a key component of the AR transcriptional complex yet its role in prostate cancer progression and the relationship between AR and FOXA1 are not completely resolved. It is well established that FOXA1 levels are elevated in advanced prostate cancer and metastases. We mimicked these conditions by overexpressing FOXA1 in the androgen-responsive LNCaP prostate cancer cell line and observed a significant increase in AR genomic binding at novel regions that possess increased chromatin accessibility. High levels of FOXA1 resulted in increased proliferation at both sub-optimal and high 5α-dihydrotestosterone (DHT) concentrations. Immunohistochemical staining for FOXA1 in a clinical prostate cancer cohort revealed that high FOXA1 expression is associated with shorter time to biochemical recurrence after radical prostatectomy (hazard ratio (HR) 5.0, 95% confidence interval (CI) 1.2-21.1, P=0.028), positive surgical margins and higher stage disease at diagnosis. The gene expression program that results from FOXA1 overexpression is enriched for PTEN, Wnt and other pathways typically represented in CRPC gene signatures. Together, these results suggest that in an androgen-depleted state, elevated levels of FOXA1 enhance AR binding at genomic regions not normally occupied by AR, which in turn facilitates prostate cancer cell growth.

Minireview: The androgen receptor in breast tissues: growth inhibitor, tumor suppressor, oncogene?

Androgen receptor (AR) signaling exerts an antiestrogenic, growth-inhibitory influence in normal breast tissue, and this role may be sustained in estrogen receptor α (ERα)-positive luminal breast cancers. Conversely, AR signaling may promote growth of a subset of ERα-negative, AR-positive breast cancers with a molecular apocrine phenotype. Understanding the molecular mechanisms whereby androgens can elicit distinct gene expression programs and opposing proliferative responses in these two breast cancer phenotypes is critical to the development of new therapeutic strategies to target the AR in breast cancer.

Evaluation of surgical experience and the use of an osteotomy guide on the apical angle of an Austin osteotomy.

BACKGROUND: Distal osteotomies of the first metatarsal are commonly used to correct hallux valgus deformities. Of the distal osteotomies, the Austin osteotomy is popular among foot surgeons on an international level. The precision of the osteotomy is important to achieve a congruous osteotomy. OBJECTIVES: The purpose of this study was to examine the effects of experience and technique on creating a precise Austin osteotomy. METHOD: Three individuals with varying levels of experience (student, resident and podiatric physician) created Austin osteotomies in metatarsal sawbones, using three different techniques (freehand, guide wire and osteotomy guide). The medial and lateral apical angles were measured, and the mean, standard deviation, and range of the angles were calculated. The differences between medial and lateral angles were also calculated. RESULTS: The results indicated that the mean and range of the angles varied considerably with the freehand and guide wire techniques at all experience levels. The angles were accurate and consistent for all experience levels; however, when an osteotomy guide was used. The use of an osteotomy guide also noticeably reduced the number of divergent and convergent osteotomies. CONCLUSIONS: The use of an osteotomy guide consistently resulted in a more precise Austin osteotomy for all experience levels.

Design of high reliability organizations in health care.

To improve safety performance, many healthcare organizations have sought to emulate high reliability organizations from industries such as nuclear power, chemical processing, and military operations. We outline high reliability design principles for healthcare organizations including both the formal structures and the informal practices that complement those structures. A stage model of organizational structures and practices, moving from local autonomy to formal controls to open inquiry to deep self-understanding, is used to illustrate typical challenges and design possibilities at each stage. We suggest how organizations can use the concepts and examples presented to increase their capacity to self-design for safety and reliability.

Redirecting traditional professional values to support safety: changing organisational culture in health care.

Professionals in healthcare organisations who seek to enhance safety and quality in an increasingly demanding industry environment often identify culture as a barrier to change. The cultural focus on individual autonomy, for example, seems to conflict with desired norms of teamwork, problem reporting, and learning. We offer a definition and explication of why culture is important to change efforts. A cultural analysis of health care suggests professional values that can be redirected to support change. We offer examples of organisations that drew upon cultural strengths to create new ways of working and gradually shifted the culture.

Estrogen and antiestrogen regulation of cell cycle progression in breast cancer cells.

The central involvement of estrogen in the development of the mammary gland and in the genesis of breast cancer has lent impetus to studies of the links between estrogen action and the cell cycle machinery. Recent studies of the estrogenic regulation of molecules with known roles in the control of G1/S phase progression have resulted in significant advances in understanding these links. Estrogens independently regulate the expression and function of c-Myc and cyclin D1 and the induction of either c-Myc or cyclin D1 is sufficient to recapitulate the effects of estrogen on cell cycle progression. These pathways converge at the activation of cyclin E-Cdk2 complexes. The active cyclin E-Cdk2 complexes are depleted of the cyclin dependent kinase (CDK) inhibitor p21(WAF1/CIP1) because of estrogen-mediated inhibition of nascent p21(WAF1/CIP1). Insulin and estrogen synergistically stimulate cell cycle progression, and the ability of estrogen to antagonize an insulin-induced increase in p21(WAF1/CIP1) gene expression appears to underlie this effect. Antiestrogen treatment of MCF-7 cells leads to an acute decrease of c-Myc expression, a subsequent decline in cyclin D1, and ultimately arrest of cells in a state with features characteristic of quiescence. An antisense-mediated decrease in c-Myc expression results in decreased cyclin D1 expression and inhibition of DNA synthesis, mimicking the effects of antiestrogen treatment and emphasizing the importance of c-Myc as an estrogen/antiestrogen target. These data identify c-Myc, cyclin D1, p21(WAF1/CIP1) and cyclin E-Cdk2 as central components of estrogen regulation of cell cycle progression and hence as potential downstream targets that contribute to the role of estrogen in oncogenesis.

Lessons learned from non-medical industries: root cause analysis as culture change at a chemical plant.

Root cause analysis was introduced to a chemical plant as a way of enhancing performance and safety, exemplified by the investigation of an explosion. The cultural legacy of the root cause learning intervention was embodied in managers' increased openness to new ideas, individuals' questioning attitude and disciplined thinking, and a root cause analysis process that provided continual opportunities to learn and improve. Lessons for health care are discussed, taking account of differences between the chemical and healthcare industries.

Leading organisational learning in health care.

As healthcare organisations seek to enhance safety and quality in a changing environment, organisational learning practices can help to improve existing skills and knowledge and provide opportunities to discover better ways of working together. Leadership at executive, middle management, and local levels is needed to create a sense of shared purpose. This shared vision should help to build effective relationships, facilitate connections between action and reflection, and strengthen the desirable elements of the healthcare culture while modifying outdated assumptions, procedures, and structures.

A low abundance pool of nascent p21WAF1/Cip1 is targeted by estrogen to activate cyclin E*Cdk2.

Estrogens regulate cell proliferation in target tissues, including breast cancer by stimulating G(1)-S phase transition. Activation of cyclin E.Cdk2 through abrogation of the ability of p21(WAF1/Cip1) to bind to and inhibit cyclin-CDKs is a pivotal event in this process in MCF-7 breast cancer cells. A proposed mechanism is p21 sequestration into cyclin D1.Cdk4/6 complexes driven by estrogen-induced transcriptional activation of cyclin D1 gene expression. However, we now show that some E(2)-induced cyclin E.Cdk2 activation occurs in the absence of increased cyclin D1 levels and requires decreased p21 protein synthesis. Both mechanisms operate in the absence of major changes in total p21 protein levels and instead target a low abundance subset of newly synthesized p21. E(2)-induced activation of cyclin E.Cdk2 is mimicked by targeted inhibition of nascent p21 expression by antisense p21 oligonucleotides. Cyclin E.Cdk2 activation is completely inhibited by a combination of antisense cyclin D1 oligonucleotide transfection and elimination of the decrease in nascent p21 by infection with adenoviral-p21. These findings strongly support a central role for p21 in the early phase of E(2)-induced mitogenesis and highlight a major functional role for newly synthesized CDK inhibitory proteins.

Digitalis-like factor response to hyperinsulinemia accompanying a euglycemic hyperinsulinemic clamp or oral glucose tolerance test.

Many studies of essential hypertension find evidence of insulin resistance in the same individuals, leading some to postulate a hypertensive role for insulin. However, the mechanisms by which insulin might exert a hypertensive effect are not fully resolved. An endogenous sodium pump inhibitor or digitalis-like factor (DLF) has been proposed as a hypertensive agent and its plasma concentrations are elevated in hypertension and in Type II diabetes, where insulin levels are elevated. Hence, we studied the effect of insulin on DLF using two approaches to achieve hyperinsulinemia. Normotensive men and women underwent a hyperinsulinemic, euglycemic clamp (40 mU/m2/min insulin, 40 mU = 1.6 x 10(-6) g) in which plasma insulin concentration was kept at high, but physiologic levels. Serum DLF (measured as inhibition of [Na,K]ATPase activity) and insulin levels were measured at baseline and every 30 min throughout the 2 hr clamp. Additionally, other subjects underwent an oral glucose tolerance test (OGTT) as a second means of increasing insulin levels. Insulin and DLF levels were measured prior to and hourly for 3 hours after receiving 100 gm of oral glucose. Serum DLF increased significantly during the clamp from a baseline of 4.6 +/- 0.81 to a peak of 8.7 +/- 1.2% inhibition (p=0.001). Comparison of the baseline and peak DLF levels with concomitant plasma insulin levels revealed a significant correlation (R=0.60, p=0.003). During the OGTT, DLF levels rose from a baseline of 2.4 +/- 1.0 to a peak level of 5.0 +/- 0.4%, p = 0.04. These results suggest that DLF, a factor that can cause vascular smooth muscle contraction and potentially influence blood pressure, is increased by hyperinsulinemia and provides a mechanism by which insulin may increase blood pressure.

Translation and validation of the Spanish version of the RELATE questionnaire using a modified serial approach for cross-cultural translation.

This article describes the initial translation and validation of the Spanish version of the RELATionshhip Evaluation (RELATE) questionnaire with a sample of monolingual English speakers (n = 78), a sample of monolingual Spanish speakers (n = 18), and two samples of Spanish/English Bilinguals (n = 27 and n = 34). Cross-cultural and cross-language equivalence of the Spanish version of RELATE to the original English version were assessed using a Modified Serial Approach (MSA) for instrument translation. Face and content validity of the Spanish RELATE were established. Test-retest reliability indices obtained with the translated version among the monolingual and bilingual Spanish speaking groups were consistently equivalent to, and in some cases higher than, the baseline reliability obtained with the monolingual English speaking group. Applications of the Spanish version of RELATE and use of the MSA for researchers and practitioners are presented.

Correlation between the glucose clamp technique and the homeostasis model assessment in hypertension.

The homeostasis model assessment (HOMA) has been extensively used as a reliable surrogate marker for measuring insulin resistance in patients with diabetes mellitus and in normal subjects. Comparative data in another insulin resistance state, hypertension, is lacking. The goal of the present study was to obtain that information by testing the correlation of HOMA with the euglycemic hyperinsulinemic clamp in 36 hypertensive and 27 normotensive subjects. Clamp-derived insulin sensitivity was calculated as the glucose disposal rate over steady-state plasma insulin concentration. Homeostasis model assessment was computed using the formula (fasting glucose x fasting insulin)/22.5. There was significant correlation between the clamp and HOMA for both the hypertensive (r = -0.64, P < .0001) and normotensive subjects (r = -0.58, P = .002). The HOMA can be used reliably as a less expensive and less cumbersome alternative for measuring insulin resistance in hypertension.

The effect of droloxifene and estrogen on thyroid function in postmenopausal women.

Estrogen is known to increase serum T4-binding globulin (TBG) concentrations, thereby increasing serum total T4 concentrations. Serum free T4 concentrations, however, remain normal. Tamoxifen, a selective estrogen receptor modifier (SERM), also raises serum TBG concentrations, but whether newer SERMs with less stimulatory action on the endometrium do so is not known. We, therefore, compared the effect of droloxifene, a SERM, and conjugated equine estrogen on pituitary-thyroid function in normal postmenopausal women. Ten women were treated for 6 weeks with conjugated estrogen (Premarin), 0.625 mg/day, and droloxifene, 60 mg/day, in a double-blind crossover study with an intervening 4-week no-treatment period. We measured serum T4, T3, TBG, free T4 index, and TSH at baseline and at the end of each 6-week period. The baseline values were compared with the 6-week values using paired t tests. The mean (+/- SD) serum TBG concentrations increased significantly during both treatment periods (baseline, 1.5+/-0.4 mg/dL; conjugated estrogens, 2.7+/-0.6 mg/dL; droloxifene, 2.1+/-0.6 mg/dL; P < 0.001 and P = 0.001, respectively). There were no significant changes in the serum free T4 index. Serum T4 and T3 concentrations increased during both treatment periods, however, the increase was significant only for T4 during the conjugated estrogen treatment period. The serum TSH concentrations increased significantly during both treatment periods (18% during conjugated estrogen and 11% during droloxifene), and the values remained within the normal range in all women. Administration of both conjugated estrogen and droloxifene for 6 weeks increases serum TSH and TBG concentrations, but does not alter free T4 index values in postmenopausal women.

A pure estrogen antagonist inhibits cyclin E-Cdk2 activity in MCF-7 breast cancer cells and induces accumulation of p130-E2F4 complexes characteristic of quiescence.

Estrogen antagonists inhibit cell cycle progression in estrogen-responsive cells, but the molecular mechanisms are not fully defined. Antiestrogen-mediated G(0)/G(1) arrest is associated with decreased cyclin D1 gene expression, inactivation of cyclin D1-cyclin dependent kinase (Cdk) 4 complexes, and decreased phosphorylation of the retinoblastoma protein (pRb). We now show that treatment of MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 results in inhibition of cyclin E-Cdk2 activity prior to a decrease in the G(1) to S phase transition. This decrease was dependent on p21(WAF1/Cip1) since treatment with antisense oligonucleotides to p21 attenuated the effect. Recruitment of p21 to cyclin E-Cdk2 complexes was in turn dependent on decreased cyclin D1 expression since it was apparent following treatment with antisense cyclin D1 oligonucleotides. To define where within the G(0) to S phase continuum antiestrogen-treated cells arrested, we assessed the relative abundance and phosphorylation state of pocket protein-E2F complexes. While both pRb and p107 levels were significantly decreased, p130 was increased 4-fold and was accompanied by the formation of p130.E2F4 complexes and the accumulation of hyperphophorylated E2F4, putative markers of cellular quiescence. Thus, ICI 182780 inhibits both cyclin D1-Cdk4 and cyclin E-Cdk2 activity, resulting in the arrest of MCF-7 cells in a state with characteristics of quiescence (G(0)), as opposed to G(1) arrest.