The AF-2 cofactor binding region is key for the selective SUMOylation of estrogen receptor alpha by antiestrogens.

Antiestrogens (AEs) are used to treat all stages of estrogen receptor (ER)-positive breast cancer. Selective estrogen receptor modulators such as tamoxifen have tissue-specific partial agonist activity, while selective estrogen receptor downregulators such as fulvestrant (ICI182,780) display a more complete antiestrogenic profile. We have previously observed that fulvestrant-induced ERα SUMOylation contributes to transcriptional suppression, but whether this effect is seen with other AEs and is specific to ERα is unclear. Here we show that several AEs induce SUMOylation of ERα, but not ERß, at different levels. Swapping domains between ERα and ERß indicates that the ERα identity of the ligand-binding domain helices 3 and 4 (H3-H4 region), which contribute to the static part of the activation function-2 (AF-2) cofactor binding groove, is sufficient to confer fulvestrant-induced SUMOylation to ERß. This region does not contain lysine residues unique to ERα, suggesting that ERα-specific residues in H3-H4 determine the capacity of the AE-bound ERα ligand-binding domain to recruit the SUMOylation machinery. We also show that the SUMO E3 ligase protein inhibitor of activated STAT 1 increases SUMOylation of ERα and of ERß containing the H3-H4 region of ERα, but not of ERß. Together, these results shed new light on the molecular basis for the differential capacity of selective estrogen receptor modulators and selective estrogen receptor downregulators to suppress transcription by ERα.

Dual-function antiandrogen/HDACi hybrids based on enzalutamide and entinostat.

The combination of androgen receptor antagonists with histone deacetylase inhibitors (HDACi) has been shown to be more effective than antiandrogens alone in halting growth of prostate cancer cell lines. Here we have designed, synthesized and assessed a series of antiandrogen/HDACi hybrids by combining structural features of enzalutamide with either SAHA or entinostat. The hybrids are demonstrated to maintain bifunctionality using a fluorometric HDAC assay and a bioluminescence resonance energy transfer (BRET) antiandrogen assay. Antiproliferative assays showed that hybrids bearing o-aminoanilide-based HDACi motifs outperformed hydroxamic acid based HDACi's. The hybrids demonstrated selectivity for epithelial cell lines vs. stromal cell lines, suggesting a potentially useful therapeutic window.

Estrogens desensitize MCF-7 breast cancer cells to apelin-induced autophagy and enhanced growth under estrogen starvation: a possible implication in endocrine resistance.

Apelin-13 is an adipokine known for its growth-inducing effects on human breast cancer cells in an estrogen-containing environment. However, the response of these cells to apelin-13 in the absence of estrogen and its association with the expression of the apelin receptor (APLNR) has not yet been investigated. In the present study, we show that the breast cancer cell line MCF-7 expresses the APLNR as shown by immunofluorescence and flow cytometry, under conditions of ER starvation and that culture of these cells in the presence of apelin-13 results in an increased growth rate and a diminished autophagy flux.  Moreover, the binding of APLNR by apelin-13 resulted in an increased growth rate (assayed by AlamarBlue) and a diminished autophagy flux (monitored by Lysotracker Green). The latter observations were reversed in the presence of exogenous estrogen. Finally, apelin-13 induces the deactivation of the apoptotic kinase AMPK. Taken together, our results show that APLNR signaling in breast cancer cells is functional and prevents tumor growth under conditions of estrogen starvation. They furthermore suggest an alternative mechanism of estrogen-independent tumor growth thereby positioning the APLNR-AMPK axis as a novel pathway and a possible therapeutic target in endocrine resistance of breast cancer cells.

Monitoring ligand-dependent assembly of receptor ternary complexes in live cells by BRETFect.

There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of ternary complexes in live cells. Here we describe bioluminescence resonance energy transfer with fluorescence enhancement by combined transfer (BRETFect), a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate. Its unique donor-intermediate-acceptor relay system is designed so that the acceptor can receive energy either directly from the donor or indirectly via the intermediate in a combined transfer, taking advantage of the entire luciferase emission spectrum. BRETFect was used to study the ligand-dependent cofactor interaction properties of the estrogen receptors ERα and ERß, which form homo- or heterodimers whose distinctive regulatory properties are difficult to dissect using traditional methods. BRETFect uncovered the relative capacities of hetero- vs. homodimers to recruit receptor-specific cofactors and regulatory proteins, and to interact with common cofactors in the presence of receptor-specific ligands. BRETFect was also used to follow the assembly of ternary complexes between the V2R vasopressin receptor and two different intracellular effectors, illustrating its use for dissection of ternary protein-protein interactions engaged by G protein-coupled receptors. Our results indicate that BRETFect represents a powerful and versatile technique to monitor the dynamics of ternary interactions within multimeric complexes in live cells.

Single-cell profiling reveals that eRNA accumulation at enhancer-promoter loops is not required to sustain transcription.

Enhancers are intergenic DNA elements that regulate the transcription of target genes in response to signaling pathways by interacting with promoters over large genomic distances. Recent studies have revealed that enhancers are bi-directionally transcribed into enhancer RNAs (eRNAs). Using single-molecule fluorescence in situ hybridization (smFISH), we investigated the eRNA-mediated regulation of transcription during estrogen induction in MCF-7 cells. We demonstrate that eRNAs are localized exclusively in the nucleus and are induced with similar kinetics as target mRNAs. However, eRNAs are mostly nascent at enhancers and their steady-state levels remain lower than those of their cognate mRNAs. Surprisingly, at the single-allele level, eRNAs are rarely co-expressed with their target loci, demonstrating that active gene transcription does not require the continuous transcription of eRNAs or their accumulation at enhancers. When co-expressed, sub-diffraction distance measurements between nascent mRNA and eRNA signals reveal that co-transcription of eRNAs and mRNAs rarely occurs within closed enhancer-promoter loops. Lastly, basal eRNA transcription at enhancers, but not E2-induced transcription, is maintained upon depletion of MLL1 and ERα, suggesting some degree of chromatin accessibility prior to signal-dependent activation of transcription. Together, our findings suggest that eRNA accumulation at enhancer-promoter loops is not required to sustain target gene transcription.

MiSTIC, an integrated platform for the analysis of heterogeneity in large tumour transcriptome datasets.

Genome-wide transcriptome profiling has enabled non-supervised classification of tumours, revealing different sub-groups characterized by specific gene expression features. However, the biological significance of these subtypes remains for the most part unclear. We describe herein an interactive platform, Minimum Spanning Trees Inferred Clustering (MiSTIC), that integrates the direct visualization and comparison of the gene correlation structure between datasets, the analysis of the molecular causes underlying co-variations in gene expression in cancer samples, and the clinical annotation of tumour sets defined by the combined expression of selected biomarkers. We have used MiSTIC to highlight the roles of specific transcription factors in breast cancer subtype specification, to compare the aspects of tumour heterogeneity targeted by different prognostic signatures, and to highlight biomarker interactions in AML. A version of MiSTIC preloaded with datasets described herein can be accessed through a public web server (http://mistic.iric.ca); in addition, the MiSTIC software package can be obtained (github.com/iric-soft/MiSTIC) for local use with personalized datasets.

Incorporation of histone deacetylase inhibitory activity into the core of tamoxifen - A new hybrid design paradigm.

Hybrid antiestrogen/histone deacetylase (HDAC) inhibitors were designed by appending zinc binding groups to the 4-hydroxystilbene core of 4-hydroxytamoxifen. The resulting hybrids were fully bifunctional, and displayed high nanomolar to low micromolar IC50 values against both the estrogen receptor α (ERα) and HDACs in vitro and in cell-based assays. The hybrids were antiproliferative against ER+ MCF-7 breast cancer cells, with hybrid 28b possessing an improved activity profile compared to either 4-hydroxytamoxifen or SAHA. Hybrid 28b displayed gene expression patterns that reflected both ERα and HDAC inhibition.

Alu repeats as transcriptional regulatory platforms in macrophage responses to M. tuberculosis infection.

To understand the epigenetic regulation of transcriptional response of macrophages during early-stage M. tuberculosis (Mtb) infection, we performed ChIPseq analysis of H3K4 monomethylation (H3K4me1), a marker of poised or active enhancers. De novo H3K4me1 peaks in infected cells were associated with genes implicated in host defenses and apoptosis. Our analysis revealed that 40% of de novo regions contained human/primate-specific Alu transposable elements, enriched in the AluJ and S subtypes. These contained several transcription factor binding sites, including those for members of the MEF2 and ATF families, and LXR and RAR nuclear receptors, all of which have been implicated in macrophage differentiation, survival, and responses to stress and infection. Combining bioinformatics, molecular genetics, and biochemical approaches, we linked genes adjacent to H3K4me1-associated Alu repeats to macrophage metabolic responses against Mtb infection. In particular, we show that LXRα signaling, which reduced Mtb viability 18-fold by altering cholesterol metabolism and enhancing macrophage apoptosis, can be initiated at response elements present in Alu repeats. These studies decipher the mechanism of early macrophage transcriptional responses to Mtb, highlighting the role of Alu element transposition in shaping human transcription programs during innate immunity.

MCM2: An alternative to Ki-67 for measuring breast cancer cell proliferation.

Breast cancer is a heterogeneous disease comprising a diversity of tumor subtypes that manifest themselves in a wide variety of clinical, pathological, and molecular features. One important subset, luminal breast cancers, comprises two clinically distinct subtypes luminal A and B each of them endowed with its own genetic program of differentiation and proliferation. Luminal breast cancers were operationally defined as follows: Luminal A: ER+, PR+, HER2-, Ki-67<14% and Luminal B: ER+ and/or PR+, HER2-,Ki-67≥14% or, alternatively ER+ and/or PR+, HER2+, any Ki-67. There is currently a need for a clinically robust and validated immunohistochemical assay that can help distinguish between luminal A and B breast cancer. MCM2 is a family member of the minichromosome maintenance protein complex whose role in DNA replication and cell proliferation is firmly established. As MCM2 appears to be an attractive alternative to Ki-67, we sought to study the expression of MCM2 and Ki-67 in different histological grades and molecular subtypes of breast cancer focusing primarily on ER-positive tumors. MCM2 and Ki-67 mRNA expression were studied using in silico analysis of available DNA microarray and RNA-sequencing data of human breast cancer. We next used immunohistochemistry to evaluate protein expression of MCM2 and Ki-67 on tissue microarrays of invasive breast carcinoma. We found that MCM2 and Ki-67 are highly expressed in breast tumors of high histological grades, comprising clinically aggressive tumors such as triple-negative, HER2-positive and luminal B subtypes. MCM2 expression was detected at higher levels than that of Ki-67 in normal breast tissues and in breast cancers. The bimodal distribution of MCM2 scores in ER+/HER2- breast tumors led to the identification of two distinct subgroups with different relapse-free survival rates. In conclusion, MCM2 expression can help sorting out two clinically important subsets of luminal breast cancer whose treatment and clinical outcomes are likely to diverge.

Selective ligand activity at Nur/retinoid X receptor complexes revealed by dimer-specific bioluminescence resonance energy transfer-based sensors.

Retinoid X receptors (RXRs) play a role as master regulators because of their capacity to form heterodimers with other nuclear receptors (NRs). Accordingly, retinoid signaling is involved in multiple biologic processes, including development, cell differentiation, metabolism, and cell death. However, the role and function of RXRs in different heterodimer complexes remain unidentified, mainly because most RXR drugs (called rexinoids) are not selective of specific heterodimer complexes. The lack of selectivity strongly limits the use of rexinoids for specific therapeutic approaches. To better characterize rexinoids at specific NR complexes, we have developed and optimized luciferase (Luc) protein complementation(PCA)-based bioluminescence resonance energy transfer (BRET) assays that can directly measure recruitment of a coactivator (CoA) motif fused to yellow fluorescent protein (YFP) by specific NR dimers. To validate the assays, we compared rexinoid modulation of CoA recruitment by the RXR homodimer and by the heterodimers Nur77/RXR and Nurr1/RXR. Results revealed that some rexinoids display selective CoA recruitment activities with homo- or heterodimer complexes. In particular, SR11237 (BMS649) has stronger potency for recruitment of CoA motif and transcriptional activity with the heterodimer Nur77/RXR than other complexes. This technology should be useful in identifying new compounds with specificity for individual dimeric species formed by NRs.

Ligand-dependent corepressor contributes to transcriptional repression by C2H2 zinc-finger transcription factor ZBRK1 through association with KRAB-associated protein-1.

We identified a novel interaction between ligand-dependent corepressor (LCoR) and the corepressor KRAB-associated protein-1 (KAP-1). The two form a complex with C2H2 zinc-finger transcription factor ZBRK1 on an intronic binding site in the growth arrest and DNA-damage-inducible α (GADD45A) gene and a novel site in the fibroblast growth factor 2 (FGF2) gene. Chromatin at both sites is enriched for histone methyltransferase SETDB1 and histone 3 lysine 9 trimethylation, a repressive epigenetic mark. Depletion of ZBRK1, KAP-1 or LCoR led to elevated GADD45A and FGF2 expression in malignant and non-malignant breast epithelial cells, and caused apoptotic death. Loss of viability could be rescued by simultaneous knockdowns of FGF2 and transcriptional coregulators or by blocking FGF2 function. FGF2 was not concurrently expressed with any of the transcriptional coregulators in breast malignancies, suggesting an inverse correlation between their expression patterns. We propose that ZBRK1, KAP-1 and LCoR form a transcriptional complex that silences gene expression, in particular FGF2, which maintains breast cell viability. Given the broad expression patterns of both LCoR and KAP-1 during development and in the adult, this complex may have several regulatory functions that extend beyond cell survival, mediated by interactions with ZBRK1 or other C2H2 zinc-finger proteins.

Design, synthesis and evaluation of antiestrogen and histone deacetylase inhibitor molecular hybrids.

The combination of antiestrogens and histone deacetylase inhibitors (HDACi) has been found to be antiproliferative in breast cancer models. We designed and synthesized hybrid structures which combined structural features of the pure antiestrogen ICI-164,384 and HDACi's SAHA and entinostat in a single bifunctional molecule. The hybrids retained antiestrogenic and HDACi activity and, in the case of benzamide hybrids, were selective for Class I HDAC3 over Class II HDAC6. The hybrids possessed low micromolar to high nanomolar activity against both ER+ MCF-7 and ER- MDA-MB-231 breast cancer cell models.

LKB1 when associated with methylatedERα is a marker of bad prognosis in breast cancer.

Although the presence of nuclear estrogen receptor is widely used to guide breast cancer therapy, less attention has been paid to the receptor cytoplasmic signaling. Recently, we have shown that this pathway is operative in vivo and is activated in aggressive tumors representing a new potential target for breast cancer therapy. Here, we identified LKB1 as a partner of ERα and we explored its potential role in estrogen nongenomic signaling. The associations between LKB1 expression and the actors of this pathway, namely the methylated form of ERα (metERα), Src and PI3K, have been analyzed both in cultured cells and in 154 primary breast tumor samples. We found that LKB1 is a component of the cytoplasmic signaling complex in breast cell lines as well as in primary breast tumors. Moreover, an inverse correlation between the localization of LKB1 in nuclear and cytoplasmic compartments is observed. Importantly, high expression of cytoplasmic LKB1 is an independent marker of poor prognosis, associated with reduced overall survival (OS) and disease free survival (DFS). Conversely, the presence of nuclear LKB1 associates with increased OS and DFS. In conclusion, our results highlight that LKB1 expression in breast cancer appears to have opposite effects depending on its subcellular localization and may be used as a new prognostic biomarker.

Design, synthesis and antiproliferative activity of raloxifene/histone deacetylase inhibitor hybrids in breast cancer.

Antiestrogen/histone deacetylase inhibitor (HDACi) hybrids were designed by merging structures of raloxifene with suberoylanilide hydroxamic acid, incorporating the HDACi unit into the phenolic ring of the antiestrogen. These hybrids were synthesized with a range of HDACi chain lengths and assessed for bifunctionality. Four hybrids, 21 (YW471), 22 (YW490), 27(YW486), and 28 (YW487) showed good potency both as antiestrogens in a BRET assay and in a fluorometric HDACi assay. The antiproliferative activity of the hybrids was demonstrated in both ER+ MCF7 and ER- MDA-MB-231 breast cancer cell lines.

Deletion of immunoproteasome subunits imprints on the transcriptome and has a broad impact on peptides presented by major histocompatibility complex I molecules.

Proteasome-mediated proteolysis plays a crucial role in many basic cellular processes. In addition to constitutive proteasomes (CPs), which are found in all eukaryotes, jawed vertebrates also express immunoproteasomes (IPs). Evidence suggests that the key role of IPs may hinge on their impact on the repertoire of peptides associated to major histocompatibility complex (MHC) I molecules. Using a label-free quantitative proteomics approach, we identified 417 peptides presented by MHC I molecules on primary mouse dendritic cells (DCs). By comparing MHC I-associated peptides (MIPs) eluted from primary DCs and thymocytes, we found that the MIP repertoire concealed a cell type-specific signature correlating with cell function. Notably, mass spectrometry analyses of DCs expressing or not IP subunits MECL1 and LMP7 showed that IPs substantially increase the abundance and diversity of MIPs. Bioinformatic analyses provided evidence that proteasomes harboring LMP7 and MECL1 have specific cleavage preferences and recognize unstructured protein regions. Moreover, while differences in MIP repertoire cannot be attributed to potential effects of IPs on gene transcription, IP subunits deficiency altered mRNA levels of a set of genes controlling DC function. Regulated genes segregated in clusters that were enriched in chromosomes 4 and 8. Our peptidomic studies performed on untransfected primary cells provide a detailed account of the MHC I-associated immune self. This work uncovers the dramatic impact of IP subunits MECL1 and LMP7 on the MIP repertoire and their non-redundant influence on expression of immune-related genes.

CAXII Is a Surrogate Marker for Luminal Breast Tumors Regulated by ER and GATA3.

Estrogen receptor alpha (ERα) expression in ~2/3 breast tumors selects patients for hormonal therapies. Tumors negative for ERα but positive for the progesterone receptor (PR, encoded by PGR) have also been candidates for ER-targeting therapies, as PR expression may reflect undetected ER activity. Conversely, PR- status in ER+ tumors predicts a worse therapeutic response. Our analysis of breast tumor transcriptome datasets, however, revealed that in tumors with lower PGR expression, the clinical PR status does not correlate accurately with the expression of ESR1 or of ER target genes, including PGR itself. We identified carbonic anhydrase 12 (CA12) as an estrogen target gene better correlated with ESR1 than PGR, reflecting CA12 regulation by both ERα and the luminal factor and upstream ESR1 regulator GATA3. Immunostaining supported strong positive correlations at the protein level with ERα and GATA3 in a cohort of 118 tumors. Most ER+PR- tumors expressed CAXII at levels similar to those of ER+PR+ tumors, consistent with observations in tumor transcriptome datasets and with active estrogenic signaling in some ER+PR- breast cancer cell lines. The few ER-PR+ tumors did not express CAXII or the other luminal markers FOXA1 and GATA3. Overall, CAXII is a luminal marker that can help interpret ER status in single ER/PR positive tumors.

Stereospecific lasofoxifene derivatives reveal the interplay between estrogen receptor alpha stability and antagonistic activity in ESR1 mutant breast cancer cells.

Chemical manipulation of estrogen receptor alpha ligand binding domain structural mobility tunes receptor lifetime and influences breast cancer therapeutic activities. Selective estrogen receptor modulators (SERMs) extend estrogen receptor alpha (ERα) cellular lifetime/accumulation. They are antagonists in the breast but agonists in the uterine epithelium and/or in bone. Selective estrogen receptor degraders/downregulators (SERDs) reduce ERα cellular lifetime/accumulation and are pure antagonists. Activating somatic ESR1 mutations Y537S and D538G enable resistance to first-line endocrine therapies. SERDs have shown significant activities in ESR1 mutant setting while few SERMs have been studied. To understand whether chemical manipulation of ERα cellular lifetime and accumulation influences antagonistic activity, we studied a series of methylpyrollidine lasofoxifene (Laso) derivatives that maintained the drug's antagonistic activities while uniquely tuning ERα cellular accumulation. These molecules were examined alongside a panel of antiestrogens in live cell assays of ERα cellular accumulation, lifetime, SUMOylation, and transcriptional antagonism. High-resolution x-ray crystal structures of WT and Y537S ERα ligand binding domain in complex with the methylated Laso derivatives or representative SERMs and SERDs show that molecules that favor a highly buried helix 12 antagonist conformation achieve the greatest transcriptional suppression activities in breast cancer cells harboring WT/Y537S ESR1. Together these results show that chemical reduction of ERα cellular lifetime is not necessarily the most crucial parameter for transcriptional antagonism in ESR1 mutated breast cancer cells. Importantly, our studies show how small chemical differences within a scaffold series can provide compounds with similar antagonistic activities, but with greatly different effects of the cellular lifetime of the ERα, which is crucial for achieving desired SERM or SERD profiles.

Direct and indirect induction by 1,25-dihydroxyvitamin D3 of the NOD2/CARD15-defensin beta2 innate immune pathway defective in Crohn disease.

Vitamin D signaling through its nuclear vitamin D receptor has emerged as a key regulator of innate immunity in humans. Here we show that hormonal vitamin D, 1,25-dihydroxyvitamin D(3), robustly stimulates expression of pattern recognition receptor NOD2/CARD15/IBD1 gene and protein in primary human monocytic and epithelial cells. The vitamin D receptor signals through distal enhancers in the NOD2 gene, whose function was validated by chromatin immunoprecipitation and chromatin conformation capture assays. A key downstream signaling consequence of NOD2 activation by agonist muramyl dipeptide is stimulation of NF-kappaB transcription factor function, which induces expression of the gene encoding antimicrobial peptide defensin beta2 (DEFB2/HBD2). Pretreatment with 1,25-dihydroxyvitamin D(3) synergistically induced NF-kappaB function and expression of genes encoding DEFB2/HBD2 and antimicrobial peptide cathelicidin in the presence of muramyl dipeptide. Importantly, this synergistic response was also seen in macrophages from a donor wild type for NOD2 but was absent in macrophages from patients with Crohn disease homozygous for non-functional NOD2 variants. These studies provide strong molecular links between vitamin D deficiency and the genetics of Crohn disease, a chronic incurable inflammatory bowel condition, as Crohn's pathogenesis is associated with attenuated NOD2 or DEFB2/HBD2 function.

Positive Regulation of Estrogen Receptor Alpha in Breast Tumorigenesis.

Estrogen receptor alpha (ERα, NR3A1) contributes through its expression in different tissues to a spectrum of physiological processes, including reproductive system development and physiology, bone mass maintenance, as well as cardiovascular and central nervous system functions. It is also one of the main drivers of tumorigenesis in breast and uterine cancer and can be targeted by several types of hormonal therapies. ERα is expressed in a subset of luminal cells corresponding to less than 10% of normal mammary epithelial cells and in over 70% of breast tumors (ER+ tumors), but the basis for its selective expression in normal or cancer tissues remains incompletely understood. The mapping of alternative promoters and regulatory elements has delineated the complex genomic structure of the ESR1 gene and shed light on the mechanistic basis for the tissue-specific regulation of ESR1 expression. However, much remains to be uncovered to better understand how ESR1 expression is regulated in breast cancer. This review recapitulates the current body of knowledge on the structure of the ESR1 gene and the complex mechanisms controlling its expression in breast tumors. In particular, we discuss the impact of genetic alterations, chromatin modifications, and enhanced expression of other luminal transcription regulators on ESR1 expression in tumor cells.

Minireview: What is Known about SUMOylation Among NR4A Family Members?

NR4A receptors, including NUR77 (NR4A1), NURR1 (NR4A2) and NOR-1 (NR4A3), form a family of nuclear receptors that act as transcription factors to regulate many physiological and pathological processes such as cell cycle and apoptosis, lipid metabolism, inflammation, carcinogenesis, vascular and neuronal functions. In the absence of known endogenous ligand modulating their physiological functions, the NR4A family remains a class of orphan receptors. However, several post-translational modifications (PTMs), including SUMOylation, have been shown to regulate the expression and/or activity of these receptors. Addition of Small Ubiquitin-like MOdifier (SUMO) proteins is a dynamic and reversible enzymatic process that regulates multiple essential functions of proteins, including nuclear receptors. This review aims at summarizing what is known about the impact of SUMOylation on NR4A family member transcriptional activities and physiological functions.