ABSTRACT
Dual bromodomain BET inhibitors that bind with similar affinities to the first and second bromodomains across BRD2, BRD3, BRD4, and BRDT have displayed modest activity as monotherapy in clinical trials. Thrombocytopenia, closely followed by symptoms characteristic of gastrointestinal toxicity, have presented as dose-limiting adverse events that may have prevented escalation to higher dose levels required for more robust efficacy. ABBV-744 is a highly selective inhibitor for the second bromodomain of the four BET family proteins. In contrast to the broad antiproliferative activities observed with dual bromodomain BET inhibitors, ABBV-744 displayed significant antiproliferative activities largely although not exclusively in cancer cell lines derived from acute myeloid leukemia and androgen receptor positive prostate cancer. Studies in acute myeloid leukemia xenograft models demonstrated antitumor efficacy for ABBV-744 that was comparable with the pan-BET inhibitor ABBV-075 but with an improved therapeutic index. Enhanced antitumor efficacy was also observed with the combination of ABBV-744 and the BCL-2 inhibitor, venetoclax compared with monotherapies of either agent alone. These results collectively support the clinical evaluation of ABBV-744 in AML (Clinical Trials.gov identifier: NCT03360006).
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Pyridines/pharmacology , Pyrroles/pharmacology , Sulfonamides/pharmacology , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Cell Proliferation , Drug Therapy, Combination , Female , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Proteins of the bromodomain and extra-terminal (BET) domain family are epigenetic readers that bind acetylated histones through their bromodomains to regulate gene transcription. Dual-bromodomain BET inhibitors (DbBi) that bind with similar affinities to the first (BD1) and second (BD2) bromodomains of BRD2, BRD3, BRD4 and BRDt have displayed modest clinical activity in monotherapy cancer trials. A reduced number of thrombocytes in the blood (thrombocytopenia) as well as symptoms of gastrointestinal toxicity are dose-limiting adverse events for some types of DbBi1-5. Given that similar haematological and gastrointestinal defects were observed after genetic silencing of Brd4 in mice6, the platelet and gastrointestinal toxicities may represent on-target activities associated with BET inhibition. The two individual bromodomains in BET family proteins may have distinct functions7-9 and different cellular phenotypes after pharmacological inhibition of one or both bromodomains have been reported10,11, suggesting that selectively targeting one of the bromodomains may result in a different efficacy and tolerability profile compared with DbBi. Available compounds that are selective to individual domains lack sufficient potency and the pharmacokinetics properties that are required for in vivo efficacy and tolerability assessment10-13. Here we carried out a medicinal chemistry campaign that led to the discovery of ABBV-744, a highly potent and selective inhibitor of the BD2 domain of BET family proteins with drug-like properties. In contrast to the broad range of cell growth inhibition induced by DbBi, the antiproliferative activity of ABBV-744 was largely, but not exclusively, restricted to cell lines of acute myeloid leukaemia and prostate cancer that expressed the full-length androgen receptor (AR). ABBV-744 retained robust activity in prostate cancer xenografts, and showed fewer platelet and gastrointestinal toxicities than the DbBi ABBV-07514. Analyses of RNA expression and chromatin immunoprecipitation followed by sequencing revealed that ABBV-744 displaced BRD4 from AR-containing super-enhancers and inhibited AR-dependent transcription, with less impact on global transcription compared with ABBV-075. These results underscore the potential value of selectively targeting the BD2 domain of BET family proteins for cancer therapy.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/chemistry , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Protein Domains/drug effects , Pyridines/pharmacology , Pyrroles/pharmacology , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Mice , Pyridines/adverse effects , Pyridines/toxicity , Pyrroles/adverse effects , Pyrroles/toxicity , Rats , Receptors, Androgen/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The development of bromodomain and extraterminal domain (BET) bromodomain inhibitors and their examination in clinical studies, particularly in oncology settings, has garnered substantial recent interest. An effort to generate novel BET bromodomain inhibitors with excellent potency and drug metabolism and pharmacokinetics (DMPK) properties was initiated based upon elaboration of a simple pyridone core. Efforts to develop a bidentate interaction with a critical asparagine residue resulted in the incorporation of a pyrrolopyridone core, which improved potency by 9-19-fold. Additional structure-activity relationship (SAR) efforts aimed both at increasing potency and improving pharmacokinetic properties led to the discovery of the clinical candidate 63 (ABBV-075/mivebresib), which demonstrates excellent potency in biochemical and cellular assays, advantageous exposures and half-life both in animal models and in humans, and in vivo efficacy in mouse models of cancer progression and inflammation.
Subject(s)
Drug Discovery , Proteins/antagonists & inhibitors , Pyridones/pharmacology , Sulfonamides/pharmacology , Animals , Cell Line, Tumor , Chromatography, High Pressure Liquid , Fluorescence Resonance Energy Transfer , Half-Life , Humans , Mass Spectrometry , Mice , Proton Magnetic Resonance Spectroscopy , Pyridones/chemistry , Pyridones/pharmacokinetics , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacokineticsABSTRACT
Ten percent to 15% of all lung cancers are small-cell lung cancer (SCLC). SCLC usually grows and metastasizes before it is diagnosed and relapses rapidly upon treatment. Unfortunately, no new targeted agent has been approved in the past 30 years for patients with SCLC. The BET (bromodomain and extraterminal) proteins bind acetylated histones and recruit protein complexes to promote transcription initiation and elongation. BET proteins have been shown to regulate expression of key genes in oncogenesis, such as MYC, CCND2, and BCL2L1 Here, we demonstrate that approximately 50% of SCLC cell lines are exquisitely sensitive to growth inhibition by the BET inhibitor, ABBV-075. The majority of these SCLC cell lines underwent apoptosis in response to ABBV-075 treatment via induction of caspase-3/7 activity. ABBV-075 enhanced the expression of proapoptotic protein BIM and downregulated antiapoptotic proteins BCL2 and BCLxl to a lesser extent. Furthermore, BET inhibition increased BCL2-BIM complex, thus priming the cells for apoptosis. Indeed, strong synergy was observed both in vitro and in vivo when cotreating the cells with BET inhibitor and the BH3-mimetic, BCL2 inhibitor venetoclax (ABT-199). ABBV-075 interaction with venetoclax positively correlated with BCL2 expression. Taken together, our studies provide a rationale for treating SCLC with BET and BCL2 inhibitors in tumors with high BCL2 protein expression. Mol Cancer Ther; 16(8); 1511-20. ©2017 AACR.
Subject(s)
Apoptosis , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Lung Neoplasms/drug therapy , Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Pyridones/therapeutic use , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/pathology , Sulfonamides/therapeutic use , Animals , Apoptosis/drug effects , Bcl-2-Like Protein 11/metabolism , Biomarkers, Tumor/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Female , Humans , Lung Neoplasms/pathology , Mice, SCID , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyridones/pharmacology , Sulfonamides/pharmacology , Treatment Outcome , Xenograft Model Antitumor Assays , bcl-X Protein/metabolismABSTRACT
ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered phase I clinical trials. Comprehensive preclinical characterization of ABBV-075 demonstrated broad activity across cell lines and tumor models, representing a variety of hematologic malignancies and solid tumor indications. In most cancer cell lines derived from solid tumors, ABBV-075 triggers prominent G1 cell-cycle arrest without extensive apoptosis. In this study, we show that ABBV-075 efficiently triggers apoptosis in acute myeloid leukemia (AML), non-Hodgkin lymphoma, and multiple myeloma cells. Apoptosis induced by ABBV-075 was mediated in part by modulation of the intrinsic apoptotic pathway, exhibiting synergy with the BCL-2 inhibitor venetoclax in preclinical models of AML. In germinal center diffuse large B-cell lymphoma, BCL-2 levels or venetoclax sensitivity predicted the apoptotic response to ABBV-075 treatment. In vivo combination studies uncovered surprising benefits of low doses of ABBV-075 coupled with bortezomib and azacitidine treatment, despite the lack of in vitro synergy between ABBV-075 and these agents. The in vitro/in vivo activities of ABBV-075 described here may serve as a useful reference to guide the development of ABBV-075 and other BET family inhibitors for cancer therapy. Cancer Res; 77(11); 2976-89. ©2017 AACR.
Subject(s)
Androgen Antagonists/therapeutic use , Pyridones/therapeutic use , Sulfonamides/therapeutic use , Androgen Antagonists/pharmacology , Apoptosis , Cell Line, Tumor , Drug Synergism , Humans , Pyridones/pharmacology , Sulfonamides/pharmacology , TransfectionABSTRACT
Competitive inhibitors of acetyl-lysine binding to the bromodomains of the BET (bromodomain and extra terminal) family are being developed for the treatment of solid and hematologic malignancies. The function of BET family member BRD4 at enhancers/superenhancers has been shown to sustain signal-dependent or pathogenic gene expression programs. Here, the hypothesis was tested that the transcription factor drivers of castration-resistant prostate cancer (CRPC) clinical progression, including the androgen receptor (AR), are critically dependent on BRD4 and thus represent a sensitive solid tumor indication for the BET inhibitor ABBV-075. DHT-stimulated transcription of AR target genes was inhibited by ABBV-075 without significant effect on AR protein expression. Furthermore, ABBV-075 disrupted DHT-stimulated recruitment of BET family member BRD4 to gene-regulatory regions cooccupied by AR, including the well-established PSA and TMPRSS2 enhancers. Persistent BET inhibition disrupted the composition and function of AR-occupied enhancers as measured by a reduction in AR and H3K27Ac ChIP signal and inhibition of enhancer RNA transcription. ABBV-075 displayed potent antiproliferative activity in multiple models of resistance to second-generation antiandrogens and inhibited the activity of the AR splice variant AR-V7 and ligand-binding domain gain-of-function mutations, F877L and L702H. ABBV-075 was also a potent inhibitor of MYC and the TMPRSS2-ETS fusion protein, important parallel transcription factor drivers of CRPC. IMPLICATIONS: The ability of BET family inhibitor ABBV-075 to inhibit transcription activation downstream of the initiating events of transcription factors like AR and TMPRSS2:ETS fusion proteins provides a promising therapeutic option for CRPC patients who have developed resistance to second-generation antiandrogens. Mol Cancer Res; 15(1); 35-44. ©2016 AACR.
Subject(s)
Prostatic Neoplasms, Castration-Resistant/pathology , Pyridones/pharmacology , Sulfonamides/pharmacology , Transcription Factors/antagonists & inhibitors , Androgen Antagonists/pharmacology , Animals , Antineoplastic Agents/pharmacology , Benzamides , Cell Line, Tumor , Cell Proliferation/drug effects , Chromatin/metabolism , Dihydrotestosterone/pharmacology , Drug Resistance, Neoplasm/drug effects , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Inhibitory Concentration 50 , Ligands , Male , Mice , Nitriles , Phenotype , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/pharmacology , Prostatic Neoplasms, Castration-Resistant/genetics , Protein Domains , Receptors, Androgen/chemistry , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , Transcription, Genetic/drug effectsABSTRACT
SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1, that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation.
Subject(s)
Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/metabolism , Hepatocytes/drug effects , Receptors, Cytoplasmic and Nuclear/metabolism , Sumoylation/drug effects , Tannins/isolation & purification , Tannins/metabolism , Animals , Cells, Cultured , Drug Evaluation, Preclinical/methods , Gene Expression Profiling , Hepatocytes/enzymology , Humans , Inhibitory Concentration 50 , Mice , Mice, SCIDABSTRACT
Sumoylation is generally considered a repressive mark for many transcription factors. However, the in vivo importance of sumoylation for any given substrate remains unclear and is questionable because the extent of sumoylation appears exceedingly low for most substrates. Here, we permanently eliminated SF-1/NR5A1 sumoylation in mice (Sf-1(K119R, K194R, or 2KR)) and found that Sf-1(2KR/2KR) mice failed to phenocopy a simple gain of SF-1 function or show elevated levels of well-established SF-1 target genes. Instead, mutant mice exhibited marked endocrine abnormalities and changes in cell fate that reflected an inappropriate activation of hedgehog signaling and other potential SUMO-sensitive targets. Furthermore, unsumoylatable SF-1 mutants activated Shh and exhibited preferential recruitment to Shh genomic elements in cells. We conclude that the sumoylation cycle greatly expands the functional capacity of transcription factors such as SF-1 and is leveraged during development to achieve cell-type-specific gene expression in multicellular organisms.
Subject(s)
Endocrine System/embryology , Endocrine System/growth & development , Hedgehog Proteins/metabolism , Signal Transduction/physiology , Steroidogenic Factor 1/metabolism , Sumoylation/physiology , Adrenal Glands/embryology , Adrenal Glands/growth & development , Animals , Animals, Newborn , Antigens, CD , Antigens, Differentiation, B-Lymphocyte , Carrier Proteins/metabolism , Cells, Cultured , Corticosterone/metabolism , Electrophoretic Mobility Shift Assay/methods , Embryo, Mammalian , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental/genetics , Hedgehog Proteins/genetics , Immunoprecipitation , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Leydig Cells/metabolism , Leydig Cells/physiology , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Transgenic , Models, Biological , Mutation/genetics , Oligonucleotide Array Sequence Analysis , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Signal Transduction/genetics , Spermatozoa/growth & development , Steroidogenic Factor 1/genetics , Sumoylation/genetics , Testis/embryology , Testis/growth & development , Testosterone/metabolism , Transfection/methods , Zinc Finger Protein GLI1ABSTRACT
Progesterone is an ovarian steroid hormone that is essential for normal breast development. The actions of progesterone are largely mediated through binding to its cognate steroid hormone receptor, the progesterone receptor (PR). PR isoforms exist in the nucleus and transcriptionally activate genes necessary for proliferation and survival (classical role). Cytoplasmic or membrane-associated PR exists in the cytoplasm where it participates in protein complexes with signaling molecules and other steroid hormone receptors capable of rapid activation of cytoplasmic protein kinase cascades. This review details the extra nuclear scaffolding actions of PR with c-Src and MEK1, the upstream components of MAP kinase modules.
Subject(s)
Cell Membrane/metabolism , MAP Kinase Signaling System , Nuclear Matrix-Associated Proteins/metabolism , Receptors, Progesterone/metabolism , Signal Transduction , Animals , HumansABSTRACT
SUMO modification of nuclear receptors, including the constitutively active receptor steroidogenic factor 1 (SF-1; NR5A1), is proposed to repress their transcriptional activity. We examined the functional and structural consequences of SF-1 sumoylation at two conserved lysines (Lys119 and Lys194) that reside adjacent to the DNA-binding domain (DBD) and ligand-binding domain (LBD), respectively. Surprisingly, while previous loss-of-function studies predicted that sumoylation at Lys194 would greatly impact SF-1 function, the conformation and coregulator recruitment of fully sumoylated SF-1 LBD protein was either unchanged or modestly impaired. Sumoylation at Lys194 also modestly reduced Ser203 phosphorylation. In contrast to these findings, sumoylation of the DBD at Lys119 resulted in a marked and selective loss of DNA binding to noncanonical SF-1 targets, such as inhibinalpha; this binding deficit was extended to all sites when the sumoylated human mutant (R92Q) protein, which exhibits lower activity, was used. Consistent with this result, the K119R mutant, compared to wild-type SF-1, was selectively recruited to a "SUMO-sensitive" site in the endogenous inhibinalpha promoter, leading to increased transcription. DNA binding and sumoylation of Lys119 appeared to be mutually exclusive, suggesting that once SF-1 is bound to DNA, sumoylation may be less important in regulating SF-1 activity. We propose that sumoylation of nuclear receptors imposes an active posttranslational mark that dampens recognition of SUMO-sensitive target genes to restrain their expression.
Subject(s)
Gene Expression Regulation , Protein Processing, Post-Translational , Small Ubiquitin-Related Modifier Proteins/metabolism , Steroidogenic Factor 1/metabolism , Amino Acid Sequence , Animals , Cell Line , DNA/metabolism , Humans , Inhibins/genetics , Inhibins/metabolism , Lysine/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Promoter Regions, Genetic , Protein Conformation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/genetics , Steroidogenic Factor 1/genetics , Transcription, GeneticABSTRACT
Human progesterone receptors (PR) rapidly activate cytosolic signaling pathways, in addition to their classical function as ligand-activated transcription factors. Using ER+/PR-B+ T47D breast cancer cells, we probed the role of progestin-stimulated rapid PR signaling in the transcriptional regulation of target genes involved in breast cancer cell proliferation. Epidermal growth factor receptor (EGFR) was rapidly activated after a 10-min treatment with R5020. Progestin induced EGFR-, c-Src-, and MAPK-dependent phosphorylation of PR-B on the MAPK consensus site, Ser345. Ser345-phosphorylated PR-B receptors strongly associated with specificity protein 1 (Sp1) transcription factors to regulate PR cell cycle (p21) and growth-promoting (EGFR) target genes whose promoters lack canonical progesterone response element sequences. Inhibitors of EGFR, c-Src, or MAPK activities blocked PR tethering to Sp1 and progestin-stimulated S-phase entry. Mutant PR-B receptors defective for c-Src binding (mPro) were not phosphorylated on Ser345 in response to progestin and failed to interact with Sp1. Hormone-induced complexes containing Sp1 and wild-type PR-B, but not S345A or mPro PR-B, were recruited to Sp1 sites within the endogenous p21 promoter. Progestin-induced S-phase entry was attenuated in T47D cells containing wild-type PR-B and treated with EGFR, c-Src, or MAPK kinase inhibitors or in T47D cells stably expressing mPro or mutant DNA-binding domain PR-B. In sum, rapid progestin-activated PR signaling leads to PR Ser345 phosphorylation and tethering to Sp1. These events are critical for progestin-stimulated regulation of Sp1 target genes and breast cancer cell proliferation. Our data demonstrate the therapeutic potential for PR-targeted breast cancer treatment by exploiting multiple nodes along the PR signaling pathway, including PR-B, EGFR, c-Src, MAPK, or Sp1.
Subject(s)
Receptors, Progesterone/metabolism , Serine/metabolism , Signal Transduction/physiology , Sp1 Transcription Factor/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Chromatin Immunoprecipitation , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Flow Cytometry , Humans , Immunoblotting , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Models, Biological , Phosphorylation/drug effects , Progestins/pharmacology , Promegestone/pharmacology , Protein Binding/drug effects , Proto-Oncogene Proteins pp60(c-src)/metabolism , Receptors, Progesterone/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/genetics , Sp1 Transcription Factor/genetics , Transcription, GeneticABSTRACT
Progesterone receptors (PRs) mediate proliferation during breast development and contribute to breast cancer progression, in part by synergizing with peptide growth factors. We have previously identified PR Ser294 as a key site for direct regulation of PR location, activity, and turnover in response to phosphorylation events. Herein, we sought to better understand how hormonal cross talk alters PR function. We demonstrate that progestins (R5020 and RU486) induce rapid (15 min) sumoylation of PR Lys388; sumoylation represses PR transcriptional activity on selected progesterone response element-driven and endogenous promoters and retards ligand-induced PR down-regulation. Consistent with this finding, we show that stabilized but weakly active phospho-mutant S294A PRs are heavily sumoylated. Conversely, desumoylated PR, created by mutation of PR Lys388 (K388R) or by overexpression of sentrin (SUMO)-specific protease desumoylating enzymes, are hypersensitive to low progestin concentrations. Combination of K388R and S294A mutations (KRSA double-mutant PR) rescues both transcription and turnover of impaired phospho-mutant (S294A) receptors. Notably, phosphorylation events antagonize PR-B but not PR-A sumoylation. Treatment of cells with epidermal growth factor or transient expression of activated mitogen-activated protein/ERK kinase kinase or cyclin-dependent protein kinase 2 induces PR-B Ser294 phosphorylation and blocks PR-B sumoylation, thereby derepressing receptor activity; PR-A is resistant to these events. Modulation of reversible PR sumoylation in response to diverse hormonal signals provides a mechanism for rapid isoform-specific changes in hormone responsiveness. In the context of elevated protein kinase activities, such as during mammary gland development or breast cancer progression, phosphorylated PR-B may be undersumoylated, transcriptionally hyperactive, and unstable/undetectable.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Receptors, Progesterone/antagonists & inhibitors , Receptors, Progesterone/metabolism , SUMO-1 Protein/metabolism , Animals , Cell Line , Chlorocebus aethiops , Down-Regulation , Endopeptidases/genetics , Endopeptidases/metabolism , Hormones/metabolism , Humans , Ligands , Lysine/genetics , Lysine/metabolism , Mutation/genetics , Phosphorylation , Promoter Regions, Genetic/genetics , Receptors, Progesterone/genetics , Sensitivity and Specificity , Transcription, Genetic/geneticsABSTRACT
Progesterone receptor (PR) ligand binding induces rapid and transient (5- to 10-min) activation of cytosolic c-Src-Ras-Erk1/2 mitogen-activated protein kinase (MAPK) signaling that is independent of PR functioning as transcription factors. Here, we have explored the integration of PR-dependent transcription and rapid signaling events in breast cancer cells. PR-B, but not PR-A, induced robust and sustained (6- to 72-h) Erk1/2 activation that was required for elevated cyclin D1 protein but not mRNA levels. Sustained Erk1/2 activation in response to progestins occurred via a novel mechanism distinct from rapid signaling initiated by PR/c-Src interactions and required the PR-B DNA-binding domain (DBD). PR/progestin upregulated epidermal growth factor receptor (EGFR) and Wnt-1. In response to PR-induced Wnt-1 signaling, matrix metalloprotease (MMP)-mediated membrane-proximal shedding of EGFR ligands transactivated EGFR and induced persistent downstream c-Src and Erk1/2 activities. T47D cell anchorage-independent growth was stimulated by progestins and blocked by inhibition of Erk1/2, c-Src, EGFR, or RNA interference of Wnt-1. Similarly, cell growth in soft agar required the PR DBD but was sensitive to disruption of PR/c-Src interactions, suggesting that both PR-B-induced rapid signaling events and nuclear actions contribute to this response. Our discovery that progestins are capable of robust autocrine activation of EGFR and sustained Erk1/2 signaling provides further support for the physiological linkage of growth factor and steroid hormone signaling. PR-B-induced sustained MAPK signaling may provide prosurvival or proliferative advantages to early breast cancer lesions.
Subject(s)
ErbB Receptors/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Proto-Oncogene Proteins pp60(c-src)/physiology , Receptors, Progesterone/physiology , Wnt1 Protein/metabolism , Breast Neoplasms , Cell Line, Tumor , Enzyme Activation , Female , Humans , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Phosphorylation , Progestins/metabolism , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Signal Transduction , Transcriptional Activation , Up-RegulationABSTRACT
Progesterone receptor (PR) action is linked to epidermal growth factor (EGF) initiated signaling pathways at multiple levels; mitogen-activated protein kinases (MAPKs) are key mediators of this important cross-talk. Herein, we probed the effects of EGF on PR function and regulation of breast cancer cell growth. EGF stimulated rapid and transient phosphorylation of PR-B Ser294 relative to persistent phosphorylation of this site induced by the synthetic progestin, R5020. EGF induced nuclear translocation and DNA binding of unliganded wild-type, but not mutant PRs containing an Ala at position 294 (S294A). However, EGF alone induced little to no PR-B transcriptional activity; S294A PR-B was transcriptionally impaired. In contrast, pretreatment of cells with EGF (30min) significantly increased the potency and efficacy of wild-type, but not S294A PR transcriptional activity in response to progestin, and enhanced ligand-dependent downregulation of wild-type but not S294A PR. Replacement of Ser294 with aspartic acid (S294D) to mimic phosphorylation at this site decreased receptor stability and, as predicted, heightened progestin-induced transcription relative to wild-type PR-B. RT-PCR demonstrated the Ser294 phosphorylation-dependence of selected PR target genes (TGFalpha and HB-EGF). Surprisingly, PR-B expressing cells growing in soft agar were highly responsive to EGF or progestin, and this was further stimulated by the combination of both hormones. Cells expressing S294A PR exhibited reduced soft agar growth, and were also sensitive to R5020 alone, but failed to respond to EGF. These results suggest that PR Ser294 is an important "sensor" for growth factor inputs that affects PR function and breast cancer cell growth in the absence of progestin or in the presence of low or "sub-threshold" progestin concentrations. PR function likely contributes to breast cancer progression when EGFR family members or their ligands are overexpressed, a condition that predicts low abundance, but highly active and nuclear PR.
Subject(s)
Breast Neoplasms/metabolism , Cell Proliferation , Epidermal Growth Factor/physiology , Progestins/physiology , Receptors, Progesterone/metabolism , Signal Transduction/physiology , Transcription, Genetic/physiology , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Ligands , PhosphorylationABSTRACT
Increased protein-tyrosine kinase activity is a prognostic indicator of decreased disease-free survival in patients with advanced breast tumors. Breast tumor kinase (Brk) is a soluble protein-tyrosine kinase overexpressed in the majority of breast cancers and also in normal skin and gut epithelium, but not in normal breast epithelial cells. Herein, we show that Brk interacts with protein kinase B/Akt, a serine/threonine kinase involved in cell growth and survival. Epidermal growth factor (EGF) treatment of human keratinocytes or Brk-transfected COS-1 cells leads to the dissociation of the Brk.Akt complex, whereas a constitutively active Brk mutant containing a point mutation at Tyr-447 (YF-Brk) failed to dissociate from Akt upon EGF treatment. In addition, Brk.Akt dissociation was blocked by the inhibition of phosphatidylinositol 3-kinase. Similar to ectopic Brk, endogenous Brk in T47D breast cancer cells was less phosphorylated upon EGF treatment, but it remained constitutively associated with Akt in the presence of EGF. Overexpression of wild-type (wt)-Brk, kinase-inactive (KM)-Brk, or YF-Brk increased the Tyr phosphorylation of multiple signaling molecules including EGF receptor. However, only wt- and YF-Brk, but not KM-Brk, induced phosphorylation of Akt and inhibited the kinase activity of Akt in unstimulated cells. Similarly, overexpression of wt- or YF-, but not KM-Brk, blocked the phosphorylation of the forkhead transcription factor, a downstream Akt target. These results suggest that Brk may function as a signaling molecule whose kinase activity normally limits the activity of Akt in unstimulated cells. Additionally, these results suggest that in breast cancer cells Brk behaves similarly to a constitutively active Brk mutant (YF-Brk) and associates with tyrosine-phosphorylated proteins in deregulated signaling complexes. Together these data provide clues to the possible proto-oncogenic and oncogenic functions of Brk.