ELF5 modulates the estrogen receptor cistrome in breast cancer.

Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endocrine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 patterns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interacting with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance

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

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

ELF5 modulates the estrogen receptor cistrome in breast cancer.

Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endocrine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 patterns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interacting with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance

A mutation in the viral sensor 2’-5’-oligoadenylate synthetase 2 causes failure of lactation

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation

The effects of inducible expression of mutant and wild type <i>Oas2</i> in T47D cells.

<p><b>(A</b>) pHUSH ProEx expression vector used to express either mutant (mt) or wild type (wt) mouse <i>Oas2</i> in T47D cells in response to doxycycline (DOX). <b>(B</b>) relative expression of mt and wt <i>Oas2</i>. <b>(C</b>) Western blot showing induction of mouse OAS2 (m) running just below endogenous human OAS2 protein, with both bands above a non-specific band (nsb). <b>(D</b>) Sensitivity of the cells lines to poly I:C (pl:C) with and without DOX induction of mt and wt <i>Oas2</i>. <b>(E</b>) Effect of mt and wt <i>Oas2</i> on adherent cell number after 72h. (<b>F</b>) Cell detachment (numbers of live cells in supernatant fraction) caused by mt <i>Oas2</i>. <b>(G</b>) Effects of mt or wt <i>Oas2</i> on replating of T47D cells in a 4 hour trypsin only replating assay after 48h of DOX. <b>(H</b>) Expression of β1 integrin (β1), E-cadherin (EC) and β-actin (βa) in response to induction of mt or wt <i>Oas2</i>. <b>(I</b>) apoptotic response to induction of mt or wt <i>Oas2</i>. Data represents the average of 7 independent experiments. <b>(J</b>) cell-cycle-phase distribution at the indicated times following induction of mt or wt <i>Oas2</i>. Data represents the average of 5 independent experiments. <i>*</i>p<0.01. ANOVA 4I and J. <b>(K)</b> <i>Oas2</i> expression in parental (p) normal mouse mammary HC11 cells or in cells constitutively expressing mt or wt <i>Oas2</i>. <b>(L)</b> Effect of wt or mt Oas2 on beta Casein in HC11s after 72 hours of prolactin (Prl) and Dexamethasone (Dex) stimulation. <b>(M)</b> Effect of mt or wt <i>Oas2</i> expression on cell death at 96 hours in HC11 cells after transient transfection. All data are representative of 3 independent experiments in response to 72h of DOX except otherwise specified. Paired t-tests 4B,E,F, G, L and M.</p

Effects of knockdown of RNASEL, IRF7 and IRF3 on the effects of inducible expression of either mutant (mt) or wild type (wt) mouse <i>Oas2</i> in T47D cells.

<p><b>(A-G)</b> Provide the context of RNase L knockdown. <b>(A-C</b>) Demonstration of Doxycycline (DOX)-inducible expression of wt or mt <i>Oas2</i> in T47D cells, and effective knock-down of RNASEL (RNaL) in mt or wt expressing T47D cells by quantitative PCR (<b>B</b>) or western blot (<b>C</b>). <b>(D)</b> Effect of the induction of mt or wt OAS2 on RNase L activity <b>(E)</b> Effects of induction of mt and wt <i>Oas2</i> expression on apoptosis. <b>(F)</b> Effects of these treatments on interferon gamma protein production. <b>(G</b>) effects of these treatments on GM-CSF production. <b>(H</b>) Demonstration of effective knockdown of IRF7. <b>(I)</b> Effects of knockdown of IRF7 on mutant or wild type <i>Oas2</i>-driven apoptosis. <b>(J)</b> Demonstration of knockdown of IRF3. <b>(K)</b> Effects of knockdown of IRF3 on mutant or wild type <i>Oas2</i>-driven apoptosis.</p

Elf5 modulates the adhesion of breast cancer cells.

<p>(A) Quantification of detached cells in cultures treated with DOX (+D) compared to no induction (−D). (B) Ability of DOX-treated cells to replate 4 h after trypsin destruction of attachment proteins, compared to untreated cells. Data are expressed as a percentage of replated untreated cells. (C) Proportion of apoptotic cells in DOX treated (grey bars) compared to untreated (black bars) T47D-ELF5-V5 cells, measured by flow cytometry using the M30 antibody. (D) Expression and activation of key cell adhesion proteins following DOX induction of ELF5-V5 expression.</p

ELF5 specifies breast cancer subtype.

<p>(A) Sub network of breast cancer subtype gene sets derived from forced ELF5 expression in MCF7 luminal breast cancer cells (inner node color) and knockdown of ELF5 expression in HCC1937 basal breast cancer cells. Node size is proportional to gene set size; thicker green lines indicate greater gene set overlap. Nodes are positioned according to similarity in gene sets. Labels in bold type indicate the functional significance of the four clusters generated, label is plain type is the gene set name. The full network is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001461#pbio.1001461.s016" target="_blank">Figure S16</a>. (B–D) expression signature analysis of the ELF5-induced changes in molecular subtype produced by ELF5 knockdown in HCC1937 cells (B), or forced ELF5 expression in MCF7 cells (C), or T47D cells (D). Bars show the indicated comparisons that produce the associated <i>p</i>-values. BS, borderline significance; NS, not significant.</p