ABSTRACT
Endocrine therapies (ET) with CDK4/6 inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of 22 ER+ breast cancer patient-derived xenografts (PDXs) demonstrated that PKMYT1, a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX organoids and xenografts, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.
ABSTRACT
Cellular responses to the steroid hormones, estrogen (E2), and progesterone (P4) are governed by their cognate receptor's transcriptional output. However, the feed-forward mechanisms that shape cell-type-specific transcriptional fulcrums for steroid receptors are unidentified. Herein, we found that a common feed-forward mechanism between GREB1 and steroid receptors regulates the differential effect of GREB1 on steroid hormones in a physiological or pathological context. In physiological (receptive) endometrium, GREB1 controls P4-responses in uterine stroma, affecting endometrial receptivity and decidualization, while not affecting E2-mediated epithelial proliferation. Of mechanism, progesterone-induced GREB1 physically interacts with the progesterone receptor, acting as a cofactor in a positive feedback mechanism to regulate P4-responsive genes. Conversely, in endometrial pathology (endometriosis), E2-induced GREB1 modulates E2-dependent gene expression to promote the growth of endometriotic lesions in mice. This differential action of GREB1 exerted by a common feed-forward mechanism with steroid receptors advances our understanding of mechanisms that underlie cell- and tissue-specific steroid hormone actions.
Subject(s)
Endometriosis , Neoplasm Proteins , Receptors, Steroid , Animals , Female , Humans , Mice , Endometriosis/genetics , Endometriosis/metabolism , Endometrium/metabolism , Estrogens/metabolism , Neoplasm Proteins/metabolism , Progesterone/metabolism , Receptors, Progesterone/genetics , Receptors, Progesterone/metabolism , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Steroids/metabolismABSTRACT
Transcriptionally active ESR1 fusions (ESR1-TAF) are a potent cause of breast cancer endocrine therapy (ET) resistance. ESR1-TAFs are not directly druggable because the C-terminal estrogen/anti-estrogen-binding domain is replaced with translocated in-frame partner gene sequences that confer constitutive transactivation. To discover alternative treatments, a mass spectrometry (MS)-based kinase inhibitor pulldown assay (KIPA) was deployed to identify druggable kinases that are upregulated by diverse ESR1-TAFs. Subsequent explorations of drug sensitivity validated RET kinase as a common therapeutic vulnerability despite remarkable ESR1-TAF C-terminal sequence and structural diversity. Organoids and xenografts from a pan-ET-resistant patient-derived xenograft model that harbors the ESR1-e6>YAP1 TAF were concordantly inhibited by the selective RET inhibitor pralsetinib to a similar extent as the CDK4/6 inhibitor palbociclib. Together, these findings provide preclinical rationale for clinical evaluation of RET inhibition for the treatment of ESR1-TAF-driven ET-resistant breast cancer. SIGNIFICANCE: Kinome analysis of ESR1 translocated and mutated breast tumors using drug bead-based mass spectrometry followed by drug-sensitivity studies nominates RET as a therapeutic target. See related commentary by Wu and Subbiah, p. 3159.
Subject(s)
Antineoplastic Agents , Breast Neoplasms , Animals , Humans , Female , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Estrogen Receptor alpha/genetics , Antineoplastic Agents/therapeutic use , Disease Models, Animal , MutationABSTRACT
Genomic analysis has recently identified multiple ESR1 gene translocations in estrogen receptor alpha-positive (ERα+) metastatic breast cancer (MBC) that encode chimeric proteins whereby the ESR1 ligand binding domain (LBD) is replaced by C-terminal sequences from many different gene partners. Here we functionally screened 15 ESR1 fusions and identified 10 that promoted estradiol-independent cell growth, motility, invasion, epithelial-to-mesenchymal transition, and resistance to fulvestrant. RNA sequencing identified a gene expression pattern specific to functionally active ESR1 gene fusions that was subsequently reduced to a diagnostic 24-gene signature. This signature was further examined in 20 ERα+ patient-derived xenografts and in 55 ERα+ MBC samples. The 24-gene signature successfully identified cases harboring ESR1 gene fusions and also accurately diagnosed the presence of activating ESR1 LBD point mutations. Therefore, the 24-gene signature represents an efficient approach to screening samples for the presence of diverse somatic ESR1 mutations and translocations that drive endocrine treatment failure in MBC. SIGNIFICANCE: This study identifies a gene signature diagnostic for functional ESR1 fusions that drive poor outcome in advanced breast cancer, which could also help guide precision medicine approaches in patients harboring ESR1 mutations.
Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Biomarkers, Tumor/genetics , Breast Neoplasms/pathology , Drug Resistance, Neoplasm , Estrogen Receptor alpha/genetics , Mutation , Oncogene Proteins, Fusion/genetics , Animals , Apoptosis , Biomarkers, Tumor/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Proliferation , Estrogen Receptor alpha/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, Nude , Oncogene Proteins, Fusion/metabolism , Prognosis , Survival Rate , Transcriptome , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Estrogen receptor alpha gene (ESR1) mutations occur frequently in ER-positive metastatic breast cancer, and confer clinical resistance to aromatase inhibitors. Expression of the ESR1 Y537S mutation induced an epithelial-mesenchymal transition (EMT) with cells exhibiting enhanced migration and invasion potential in vitro. When small subpopulations of Y537S ESR1 mutant cells were injected along with WT parental cells, tumor growth was enhanced with mutant cells becoming the predominant population in distant metastases. Y537S mutant primary xenograft tumors were resistant to the antiestrogen tamoxifen (Tam) as well as to estradiol (E2) withdrawal. Y537S ESR1 mutant primary tumors metastasized efficiently in the absence of E2; however, Tam treatment significantly inhibited metastasis to distant sites. We identified a nine-gene expression signature, which predicted clinical outcomes of ER-positive breast cancer patients, as well as breast cancer metastasis to the lung. Androgen receptor (AR) protein levels were increased in mutant models, and the AR agonist dihydrotestosterone significantly inhibited estrogen-regulated gene expression, EMT, and distant metastasis in vivo, suggesting that AR may play a role in distant metastatic progression of ESR1 mutant tumors.
Subject(s)
Breast Neoplasms/drug therapy , Estrogen Receptor alpha/genetics , Receptors, Androgen/genetics , Tamoxifen/pharmacology , Animals , Aromatase Inhibitors/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Dihydrotestosterone/pharmacology , Estradiol/metabolism , Estrogens/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mutation/genetics , Neoplasm Metastasis , Receptors, Androgen/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Patients with long-term estrogen-deprived breast cancer, after resistance to tamoxifen or aromatase inhibitors develops, can experience tumor regression when treated with estrogens. Estrogen's antitumor effect is attributed to apoptosis via the estrogen receptor (ER). Estrogen treatment can have unpleasant gynecologic and nongynecologic adverse events; thus, the development of safer estrogenic agents remains a clinical priority. Here, we study synthetic selective estrogen mimics (SEM) BMI-135 and TTC-352, and the naturally occurring estrogen estetrol (E4), which are proposed as safer estrogenic agents compared with 17ß-estradiol (E2), for the treatment of endocrine-resistant breast cancer. TTC-352 and E4 are being evaluated in breast cancer clinical trials. Cell viability assays, real-time PCR, immunoblotting, ERE DNA pulldowns, mass spectrometry, X-ray crystallography, docking and molecular dynamic simulations, live cell imaging, and Annexin V staining were conducted in 11 biologically different breast cancer models. Results were compared with the potent full agonist E2, less potent full agonist E4, the benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. We report ERα's regulation and coregulators' binding profiles with SEMs and E4 We describe TTC-352's pharmacology as a weak full agonist and antitumor molecular mechanisms. This study highlights TTC-352's benzothiophene scaffold that yields an H-bond with Glu353, which allows Asp351-to-helix 12 (H12) interaction, sealing ERα's ligand-binding domain, recruiting E2-enriched coactivators, and triggering rapid ERα-induced unfolded protein response (UPR) and apoptosis, as the basis of its anticancer properties. BPTPE's phenolic OH yields an H-Bond with Thr347, which disrupts Asp351-to-H12 interaction, delaying UPR and apoptosis and increasing clonal evolution risk.
Subject(s)
Apoptosis , Breast Neoplasms/drug therapy , Drug Resistance, Neoplasm , Estrogens/analogs & derivatives , Hormones/pharmacology , Unfolded Protein Response , Apoptosis/drug effects , Benzothiazoles/metabolism , Biomarkers, Tumor/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Survival/drug effects , DNA, Neoplasm/metabolism , Drug Resistance, Neoplasm/drug effects , Endoribonucleases/metabolism , Female , Fluorescence , Gene Expression Regulation, Neoplastic/drug effects , Hormones/chemistry , Humans , Ligands , MCF-7 Cells , Models, Biological , Molecular Dynamics Simulation , Protein Binding/drug effects , Protein Domains , Protein Serine-Threonine Kinases/metabolism , Receptors, Estrogen/chemistry , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Thermodynamics , Transcription, Genetic/drug effects , Unfolded Protein Response/drug effects , X-Box Binding Protein 1/metabolismABSTRACT
Steroid receptors activate gene transcription by recruiting coactivators to initiate transcription of their target genes. For most nuclear receptors, the ligand-dependent activation function domain-2 (AF-2) is a primary contributor to the nuclear receptor (NR) transcriptional activity. In contrast to other steroid receptors, such as ERα, the activation function of androgen receptor (AR) is largely dependent on its ligand-independent AF-1 located in its N-terminal domain (NTD). It remains unclear why AR utilizes a different AF domain from other receptors despite that NRs share similar domain organizations. Here, we present cryoelectron microscopy (cryo-EM) structures of DNA-bound full-length AR and its complex structure with key coactivators, SRC-3 and p300. AR dimerization follows a unique head-to-head and tail-to-tail manner. Unlike ERα, AR directly contacts a single SRC-3 and p300. The AR NTD is the primary site for coactivator recruitment. The structures provide a basis for understanding assembly of the AR:coactivator complex and its domain contributions for coactivator assembly and transcriptional regulation.
Subject(s)
DNA/chemistry , E1A-Associated p300 Protein/metabolism , Nuclear Receptor Coactivator 3/metabolism , Receptors, Androgen/metabolism , Cryoelectron Microscopy , DNA/metabolism , E1A-Associated p300 Protein/chemistry , HEK293 Cells , Humans , Nuclear Receptor Coactivator 3/chemistry , Nucleic Acid Conformation , Protein Conformation , Receptors, Androgen/chemistry , Receptors, Androgen/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolismABSTRACT
Our early-life environment has a profound influence on developing organs that impacts metabolic function and determines disease susceptibility across the life-course. Using a rat model for exposure to an endocrine disrupting chemical (EDC), we show that early-life chemical exposure causes metabolic dysfunction in adulthood and reprograms histone marks in the developing liver to accelerate acquisition of an adult epigenomic signature. This epigenomic reprogramming persists long after the initial exposure, but many reprogrammed genes remain transcriptionally silent with their impact on metabolism not revealed until a later life exposure to a Western-style diet. Diet-dependent metabolic disruption was largely driven by reprogramming of the Early Growth Response 1 (EGR1) transcriptome and production of metabolites in pathways linked to cholesterol, lipid and one-carbon metabolism. These findings demonstrate the importance of epigenome:environment interactions, which early in life accelerate epigenomic aging, and later in adulthood unlock metabolically restricted epigenetic reprogramming to drive metabolic dysfunction.
Subject(s)
Epigenome/genetics , Animals , DNA Methylation/drug effects , DNA Methylation/genetics , Early Growth Response Protein 1/genetics , Endocrine Disruptors/toxicity , Epigenesis, Genetic/drug effects , Epigenesis, Genetic/genetics , Epigenomics/methods , Female , Gene-Environment Interaction , Genome-Wide Association Study , Male , RatsABSTRACT
High-dose synthetic estrogen therapy was the standard treatment of advanced breast cancer for three decades until the discovery of tamoxifen. A range of substituted triphenylethylene synthetic estrogens and diethylstilbestrol were used. It is now known that low doses of estrogens can cause apoptosis in long-term estrogen deprived (LTED) breast cancer cells resistant to antiestrogens. This action of estrogen can explain the reduced breast cancer incidence in postmenopausal women over 60 who are taking conjugated equine estrogens and the beneficial effect of low-dose estrogen treatment of patients with acquired aromatase inhibitor resistance in clinical trials. To decipher the molecular mechanism of estrogens at the estrogen receptor (ER) complex by different types of estrogens-planar [17ß-estradiol (E2)] and angular triphenylethylene (TPE) derivatives-we have synthesized a small series of compounds with either no substitutions on the TPE phenyl ring containing the antiestrogenic side chain of endoxifen or a free hydroxyl. In the first week of treatment with E2 the LTED cells undergo apoptosis completely. By contrast, the test TPE derivatives act as antiestrogens with a free para-hydroxyl on the phenyl ring that contains an antiestrogenic side chain in endoxifen. This inhibits early E2-induced apoptosis if a free hydroxyl is present. No substitution at the site occupied by the antiestrogenic side chain of endoxifen results in early apoptosis similar to planar E2 The TPE compounds recruit coregulators to the ER differentially and predictably, leading to delayed apoptosis in these cells. SIGNIFICANCE STATEMENT: In this paper we investigate the role of the structure-function relationship of a panel of synthetic triphenylethylene (TPE) derivatives and a novel mechanism of estrogen-induced cell death in breast cancer, which is now clinically relevant. Our study indicates that these TPE derivatives, depending on the positioning of the hydroxyl groups, induce various conformations of the estrogen receptor's ligand-binding domain, which in turn produces differential recruitment of coregulators and subsequently different apoptotic effects on the antiestrogen-resistant breast cancer cells.
Subject(s)
Breast Neoplasms/metabolism , Estrogen Antagonists/chemical synthesis , Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/metabolism , Stilbenes/chemical synthesis , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cell Survival/drug effects , Drug Resistance, Neoplasm/drug effects , Estradiol/chemistry , Estradiol/pharmacology , Estrogen Antagonists/chemistry , Estrogen Antagonists/pharmacology , Female , Humans , MCF-7 Cells , Models, Molecular , Molecular Dynamics Simulation , Molecular Structure , Stilbenes/chemistry , Stilbenes/pharmacology , Structure-Activity RelationshipABSTRACT
We report that neurofibromin, a tumor suppressor and Ras-GAP (GTPase-activating protein), is also an estrogen receptor-α (ER) transcriptional co-repressor through leucine/isoleucine-rich motifs that are functionally independent of GAP activity. GAP activity, in turn, does not affect ER binding. Consequently, neurofibromin depletion causes estradiol hypersensitivity and tamoxifen agonism, explaining the poor prognosis associated with neurofibromin loss in endocrine therapy-treated ER+ breast cancer. Neurofibromin-deficient ER+ breast cancer cells initially retain sensitivity to selective ER degraders (SERDs). However, Ras activation does play a role in acquired SERD resistance, which can be reversed upon MEK inhibitor addition, and SERD/MEK inhibitor combinations induce tumor regression. Thus, neurofibromin is a dual repressor for both Ras and ER signaling, and co-targeting may treat neurofibromin-deficient ER+ breast tumors.
Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Estrogen Receptor alpha/genetics , Neurofibromin 1/genetics , Amino Acid Motifs , Animals , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Co-Repressor Proteins , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha/metabolism , Female , Humans , MCF-7 Cells , Mice, Nude , Mice, SCID , Mutation , Neurofibromin 1/chemistry , Neurofibromin 1/metabolism , Signal Transduction , Tamoxifen/pharmacology , Xenograft Model Antitumor Assays , ras Proteins/metabolismABSTRACT
BACKGROUND: Tributyltin (TBT) is a persistent and bioaccumulative environmental toxicant. Developmental exposure to TBT has been shown to cause fatty liver disease (steatosis), as well as increased adiposity in many species, leading to its characterization as an obesogen. OBJECTIVE: We aimed to determine the long-term effects of developmental TBT exposure on the liver. METHODS: C57BL/6J mice were exposed to a dose of TBT (0.5mg/kg body weight per day; 3.07µM) below the current developmental no observed adverse effect level (NOAEL) via drinking water, or drinking water alone, provided to the dam from preconception through lactation. Sires were exposed during breeding and lactation. Pups from two parity cycles were included in this study. Animals were followed longitudinally, and livers of offspring were analyzed by pathological evaluation, immunohistochemistry, immunoblotting, and RNA sequencing. RESULTS: Developmental exposure to TBT led to increased adiposity and hepatic steatosis at 14 and 20 weeks of age and increased liver adenomas at 45 weeks of age in male offspring. Female offspring displayed increased adiposity as compared with males, but TBT did not lead to an increase in fatty liver or tumor development in female offspring. Liver tumors in male mice were enriched in pathways and gene signatures associated with human and rodent nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). This includes down-regulation of growth hormone receptor (GHR) and of STAT5 signaling, which occurred in response to TBT exposure and preceded liver tumor development. CONCLUSIONS: These data reveal a previously unappreciated ability of TBT to increase risk for liver tumorigenesis in mice in a sex-specific manner. Taken together, these findings provide new insights into how early life environmental exposures contribute to liver disease in adulthood. https://doi.org/10.1289/EHP5414.
Subject(s)
Environmental Pollutants/toxicity , Organotin Compounds/toxicity , Adiposity , Animals , Humans , Liver Neoplasms/chemically induced , Mice , Mice, Inbred C57BL , Toxicity TestsABSTRACT
A subset of environmental chemicals acts as "obesogens" as they increase adipose mass and lipid content in livers of treated rodents. One of the most studied class of obesogens are the tin-containing chemicals that have as a central moiety tributyltin (TBT), which bind and activate two nuclear hormone receptors, Peroxisome Proliferator Activated Receptor Gamma (PPARG) and Retinoid X Receptor Alpha (RXRA), at nanomolar concentrations. Here, we have tested whether TBT chloride at such concentrations may affect the neutral lipid level in two cell line models of human liver. Indeed, using high content image analysis (HCA), TBT significantly increased neutral lipid content in a time- and concentration-dependent manner. Consistent with the observed increased lipid accumulation, RNA fluorescence in situ hybridization (RNA FISH) and RT-qPCR experiments revealed that TBT enhanced the steady-state mRNA levels of two key genes for de novo lipogenesis, the transcription factor SREBF1 and its downstream enzymatic target, FASN. Importantly, pre-treatment of cells with 2-deoxy-D-glucose reduced TBT-mediated lipid accumulation, thereby suggesting a role for active glycolysis during the process of lipid accumulation. As other RXRA binding ligands can promote RXRA protein turnover via the 26S proteasome, TBT was tested for such an effect in the two liver cell lines. We found that TBT, in a time- and dose-dependent manner, significantly reduced steady-state RXRA levels in a proteasome-dependent manner. While TBT promotes both RXRA protein turnover and lipid accumulation, we found no correlation between these two events at the single cell level, thereby suggesting an additional mechanism may be involved in TBT promotion of lipid accumulation, such as glycolysis.
Subject(s)
Down-Regulation/drug effects , Hepatocytes/drug effects , Lipid Metabolism/drug effects , Retinoid X Receptor alpha/metabolism , Trialkyltin Compounds/pharmacology , Cell Line , Deoxyglucose/pharmacology , Dose-Response Relationship, Drug , Hep G2 Cells , Hepatocytes/metabolism , Humans , In Situ Hybridization, Fluorescence , Retinoid X Receptor alpha/geneticsABSTRACT
Mining of integrated public transcriptomic and ChIP-Seq (cistromic) datasets can illuminate functions of mammalian cellular signaling pathways not yet explored in the research literature. Here, we designed a web knowledgebase, the Signaling Pathways Project (SPP), which incorporates community classifications of signaling pathway nodes (receptors, enzymes, transcription factors and co-nodes) and their cognate bioactive small molecules. We then mapped over 10,000 public transcriptomic or cistromic experiments to their pathway node or biosample of study. To enable prediction of pathway node-gene target transcriptional regulatory relationships through SPP, we generated consensus 'omics signatures, or consensomes, which ranked genes based on measures of their significant differential expression or promoter occupancy across transcriptomic or cistromic experiments mapped to a specific node family. Consensomes were validated using alignment with canonical literature knowledge, gene target-level integration of transcriptomic and cistromic data points, and in bench experiments confirming previously uncharacterized node-gene target regulatory relationships. To expose the SPP knowledgebase to researchers, a web browser interface was designed that accommodates numerous routine data mining strategies. SPP is freely accessible at https://www.signalingpathways.org .
Subject(s)
Databases, Factual , Signal Transduction , Animals , Humans , Knowledge Bases , Mammals , TranscriptomeABSTRACT
The coactivator-associated arginine methyltransferase (CARM1) functions as a regulator of transcription by methylating a diverse array of substrates. To broaden our understanding of CARM1's mechanistic actions, we sought to identify additional substrates for this enzyme. To do this, we generated CARM1 substrate motif antibodies, and used immunoprecipitation coupled with mass spectrometry to identify cellular targets of CARM1, including mediator complex subunit 12 (MED12) and the lysine methyltransferase KMT2D. Both of these proteins are implicated in enhancer function. We identified the major CARM1-mediated MED12 methylation site as arginine 1899 (R1899), which interacts with the Tudor domain-containing effector molecule, TDRD3. Chromatin immunoprecipitation-seq studies revealed that CARM1 and the methyl mark it deposits are tightly associated with ERα-specific enhancers and positively modulate transcription of estrogen-regulated genes. In addition, we showed that the methylation of MED12, at the R1899 site, and the recruitment of TDRD3 by this methylated motif are critical for the ability of MED12 to interact with activating noncoding RNAs.
ABSTRACT
Enhancers are thought to activate transcription by physically contacting promoters via looping. However, direct assays demonstrating these contacts are required to mechanistically verify such cellular determinants of enhancer function. Here, we present versatile cell-free assays to further determine the role of enhancer-promoter contacts (EPCs). We demonstrate that EPC is linked to mutually stimulatory transcription at the enhancer and promoter in vitro. SRC-3 was identified as a critical looping determinant for the estradiol-(E2)-regulated GREB1 locus. Surprisingly, the GREB1 enhancer and promoter contact two internal gene body SRC-3 binding sites, GBS1 and GBS2, which stimulate their transcription. Utilizing time-course 3C assays, we uncovered SRC-3-dependent dynamic chromatin interactions involving the enhancer, promoter, GBS1, and GBS2. Collectively, these data suggest that the enhancer and promoter remain "poised" for transcription via their contacts with GBS1 and GBS2. Upon E2 induction, GBS1 and GBS2 disengage from the enhancer, allowing direct EPC for active transcription.
Subject(s)
Breast Neoplasms/genetics , Chromatin/metabolism , Estrogens/pharmacology , Gene Expression Regulation, Neoplastic , Nuclear Receptor Coactivator 3/metabolism , Transcription, Genetic , Binding Sites , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Chromatin/genetics , Enhancer Elements, Genetic , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Female , Humans , Nuclear Receptor Coactivator 3/genetics , Promoter Regions, Genetic , Protein Binding , Tumor Cells, CulturedABSTRACT
Approximately 75% of breast cancers are estrogen receptor alpha (ERα)-positive and are treatable with endocrine therapies, but often patients develop lethal resistant disease. Frequent mutations (10-40%) in the ligand-binding domain (LBD) codons in the gene encoding ERα (ESR1) have been identified, resulting in ligand-independent, constitutively active receptors. In addition, ESR1 chromosomal translocations can occur, resulting in fusion proteins that lack the LBD and are entirely unresponsive to all endocrine treatments. Thus, identifying coactivators that bind to these mutant ERα proteins may offer new therapeutic targets for endocrine-resistant cancer. To define coactivator candidate targets, a proteomics approach was performed profiling proteins recruited to the two most common ERα LBD mutants, Y537S and D538G, and an ESR1-YAP1 fusion protein. These mutants displayed enhanced coactivator interactions as compared to unliganded wild-type ERα. Inhibition of these coactivators decreased the ability of ESR1 mutants to activate transcription and promote breast cancer growth in vitro and in vivo. Thus, we have identified specific coactivators that may be useful as targets for endocrine-resistant breast cancers.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Estrogen Receptor alpha/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Female , Humans , MCF-7 Cells , Protein Binding/genetics , Proteomics , Transcription, Genetic/genetics , Transcriptional Activation/genetics , Translocation, Genetic/geneticsABSTRACT
Alterations in both cell metabolism and transcriptional programs are hallmarks of cancer that sustain rapid proliferation and metastasis 1 . However, the mechanisms that control the interaction between metabolic reprogramming and transcriptional regulation remain unclear. Here we show that the metabolic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) regulates transcriptional reprogramming by activating the oncogenic steroid receptor coactivator-3 (SRC-3). We used a kinome-wide RNA interference-based screening method to identify potential kinases that modulate the intrinsic SRC-3 transcriptional response. PFKFB4, a regulatory enzyme that synthesizes a potent stimulator of glycolysis 2 , is found to be a robust stimulator of SRC-3 that coregulates oestrogen receptor. PFKFB4 phosphorylates SRC-3 at serine 857 and enhances its transcriptional activity, whereas either suppression of PFKFB4 or ectopic expression of a phosphorylation-deficient Ser857Ala mutant SRC-3 abolishes the SRC-3-mediated transcriptional output. Functionally, PFKFB4-driven SRC-3 activation drives glucose flux towards the pentose phosphate pathway and enables purine synthesis by transcriptionally upregulating the expression of the enzyme transketolase. In addition, the two enzymes adenosine monophosphate deaminase-1 (AMPD1) and xanthine dehydrogenase (XDH), which are involved in purine metabolism, were identified as SRC-3 targets that may or may not be directly involved in purine synthesis. Mechanistically, phosphorylation of SRC-3 at Ser857 increases its interaction with the transcription factor ATF4 by stabilizing the recruitment of SRC-3 and ATF4 to target gene promoters. Ablation of SRC-3 or PFKFB4 suppresses breast tumour growth in mice and prevents metastasis to the lung from an orthotopic setting, as does Ser857Ala-mutant SRC-3. PFKFB4 and phosphorylated SRC-3 levels are increased and correlate in oestrogen receptor-positive tumours, whereas, in patients with the basal subtype, PFKFB4 and SRC-3 drive a common protein signature that correlates with the poor survival of patients with breast cancer. These findings suggest that the Warburg pathway enzyme PFKFB4 acts as a molecular fulcrum that couples sugar metabolism to transcriptional activation by stimulating SRC-3 to promote aggressive metastatic tumours.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Glucose/metabolism , Nuclear Receptor Coactivator 3/metabolism , Phosphofructokinase-2/metabolism , Transcriptional Activation , Activating Transcription Factor 4/metabolism , Animals , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Glycolysis , Humans , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Mice , Neoplasm Metastasis , Pentose Phosphate Pathway , Phosphorylation , Phosphoserine/metabolism , Prognosis , Purines/biosynthesis , Purines/metabolism , RNA Interference , Receptors, Estrogen/metabolism , Transketolase/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Particular chromatin modifications are associated with different states of gene transcription, yet our understanding of which modifications are causal 'drivers' in promoting transcription is incomplete. Here, we discuss new developments describing the ordered, mechanistic role of select histone marks occurring during distinct steps in the RNA polymerase II (Pol II) transcription cycle. In particular, we highlight the interplay between histone marks in specifying the 'next step' of transcription. While many studies have described correlative relationships between histone marks and their occupancy at distinct gene regions, we focus on studies that elucidate clear functional consequences of specific histone marks during different stages of transcription. These recent discoveries have refined our current mechanistic understanding of how histone marks promote Pol II transcriptional progression.