ELF5 suppresses estrogen sensitivity and underpins the acquisition of antiestrogen resistance in luminal breast cancer.

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.

Feasibility and effectiveness of an evidence-based asthma service in Australian community pharmacies: a pragmatic cluster randomized trial.

OBJECTIVE: To test the feasibility, effectiveness, and sustainability of a pharmacy asthma service in primary care. METHODS: A pragmatic cluster randomized trial in community pharmacies in four Australian states/territories in 2009. Specially trained pharmacists were randomized to deliver an asthma service in two groups, providing three versus four consultations over 6 months. People with poorly controlled asthma or no recent asthma review were included. Follow-up for 12 months after service completion occurred in 30% of randomly selected completing patients. Outcomes included change in asthma control (poor and fair/good) and Asthma Control Questionnaire (ACQ) score, inhaler technique, quality of life, perceived control, adherence, asthma knowledge, and asthma action plan ownership. RESULTS: Ninety-six pharmacists enrolled 570 patients, with 398 (70%) completing. Asthma control significantly improved with both the three- and four-visit service, with no significant difference between groups (good/fair control 29% and 21% at baseline, 61% and 59% at end, p = .791). Significant improvements were also evident in the ACQ (mean change 0.56), inhaler technique (17-33% correct baseline, 57-72% end), asthma action plan ownership (19% baseline, 56% end), quality of life, adherence, perceived control, and asthma knowledge, with no significant difference between groups for any variable. Outcomes were sustained at 12 months post-service. CONCLUSIONS: The pharmacy asthma service delivered clinically important improvements in both a three-visit and four-visit service. Pharmacists were able to recruit and deliver the service with minimal intervention, suggesting it is practical to implement in practice. The three-visit service would be feasible and effective to implement, with a review at 12 months.

HSD17B4 overexpression, an independent biomarker of poor patient outcome in prostate cancer.

Steroid hormones and their metabolising enzymes have been studied extensively for their potential role in prostate cancer, with more recent interest in the androgen/estrogen inactivating enzyme 17beta-hydroxysteroid dehydrogenase type 4 (HSD17B4). Gene expression profiling showed HSD17B4 to be significantly overexpressed in prostate cancer compared to matched-benign epithelium. We therefore hypothesized that altered HSD17B4 expression may contribute to prostate cancer progression via altered hormone balance. In this study, HSD17B4 mRNA and protein expression were assessed by in situ hybridisation (ISH) and immunohistochemistry (IHC), respectively, in tissue arrays of prostate tissue from 172 patients treated by radical prostatectomy. Overexpression of HSD17B4 mRNA and protein was associated with prostate cancer (P<0.0001) and multivariate Cox proportional hazards analysis, adjusted for known prognostic indicators, demonstrated HSD17B4 mRNA and high protein expression were significant independent predictors of poor patient outcome as measured by time until PSA relapse (mRNA: hazards ratio [HR]=1.90, 95% confidence interval [CI]=1.15-3.12; P<0.0001; and protein: HR=2.09, 95% CI=1.31-3.33; P=0.0026). Here we provide strong evidence that both mRNA and protein overexpression of HSD17B4 is not only associated with the presence of prostate cancer, but is also a significant independent predictor of poor patient outcome.

Identification of downstream targets of estrogen and c-myc in breast cancer cells.

Estrogen (E) plays a pivotal regulatory role in the control of cell proliferation in the normal breast and breast cancer (BC). To identify genes with likely roles in proliferation control that are regulated by E and its downstream target c-myc, we compared transcript profiles of antiestrogens-arrested cells stimulated to reinitiate cell cycle progression by E treatment or c-myc induction. Approximately 2/3 of the probe sets significantly regulated by E (adjusted p < 0.01) increased in expression. Half of the E-regulated probe sets were also regulated by c-myc. Genes involved in cell growth, cell proliferation, and cell survival were over-represented in the E-regulated geneset. Analysis of selected candidates has identified a nucleolar protein whose expression is correlated with c-myc expression in BC cell lines. These data indicate that a significant component of E-induced mitogenesis is mediated by c-myc and that selected c-myc target genes may be surrogate markers of c-myc expression in BC.

c-Myc overexpression and endocrine resistance in breast cancer.

The oncoprotein c-Myc is frequently overexpressed in breast cancer and ectopic expression in breast cancer cell lines attenuates responses to antiestrogen treatment. Here, we review preliminary data aimed at further elucidating a potential role for c-Myc in clinical endocrine resistance in breast cancer. Immunohistochemical and semi-quantitative PCR revealed that c-Myc protein and c-myc mRNA were frequently overexpressed in both ER-positive and ER-negative breast carcinoma. Furthermore, both constitutive and inducible c-Myc overexpression in MCF-7 breast cancer cell lines markedly reduced their sensitivity to the growth inhibitory effects of the pure antiestrogen ICI 182,780. In order to identify potential downstream targets of c-Myc that mediate this effect, Affymetrix microarrays were employed to examine the patterns of gene expression shared by MCF-7 cells stimulated by estrogen, or by induction of c-Myc. Approximately 50% of estrogen target genes identified 6h after treatment were also regulated by c-Myc. One novel target, EMU4, was transcriptionally regulated by c-Myc. In addition, there was a strong correlation between c-myc and EMU4 mRNA expression in a battery of breast cancer cell lines. These data confirm that c-Myc overexpression is a common event in breast cancer, and that this is associated with resistance to antiestrogens in vitro. Furthermore, the development of an experimental paradigm for the discovery of c-Myc and estrogen target genes associated with endocrine resistance provides a framework for the discovery and validation of genes involved in estrogen signalling, and c-Myc-mediated-antiestrogen resistance.

Meta-analysis and gene set enrichment relative to er status reveal elevated activity of MYC and E2F in the "basal" breast cancer subgroup.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Identification of functional networks of estrogen- and c-Myc-responsive genes and their relationship to response to tamoxifen therapy in breast cancer.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: These functionally-based gene signatures can stratify patients treated with tamoxifen into groups with differing outcome, and potentially identify distinct mechanisms of tamoxifen resistance.

Molecular evolutionary relationships of enteroinvasive Escherichia coli and Shigella spp.

Enteroinvasive Escherichia coli (EIEC), a distinctive pathogenic form of E. coli causing dysentery, is similar in many properties to bacteria placed in the four species of Shigella. Shigella has been separated as a genus but in fact comprises several clones of E. coli. The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of Shigella strains. The EIEC strains were grouped in four clusters with one outlier strain, indicating independent derivation of EIEC several times. Three of the four clusters contain more than one O antigen type. One EIEC strain (an O112ac:H- strain) was found in Shigella cluster 3 but is not identical to the Shigella cluster 3 D2 and B15 strains with the same O antigen. Two forms of the virulence plasmid pINV have been identified in Shigella strains by using the sequences of ipgD and mxiA genes, and all but two of our EIEC strains have pINV A. The EIEC strains were grouped in two subclusters with a very low level of variation, generally not intermingled with Shigella pINV A strains. The EIEC clusters based on housekeeping genes were reflected in the plasmid gene sequences, with some exceptions. Two strains were found in the pINV B form by using the ipgD sequence, with one strain having an mxiA sequence similar to the divergent sequence of D1. Clearly, EIEC and Shigella spp. form a pathovar of E. coli.