RESUMO
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.
Assuntos
Endometriose , Proteínas de Neoplasias , Receptores de Esteroides , Animais , Feminino , Humanos , Camundongos , Endometriose/genética , Endometriose/metabolismo , Endométrio/metabolismo , Estrogênios/metabolismo , Proteínas de Neoplasias/metabolismo , Progesterona/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Esteroides/metabolismoRESUMO
Steroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were "permanently eradicated" in a genetically engineered tamoxifen-inducible Treg-cell-specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the chemokine (C-C motif) ligand (Ccl) 19/Ccl21/chemokine (C-C motif) receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-γ/C-X-C motif chemokine ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and natural killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish preestablished breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3-deleted Tregs represents an approach to completely block tumor growth and recurrence without the autoimmune side effects that typically accompany immune checkpoint modulators.
Assuntos
Neoplasias da Mama , Neoplasias Mamárias Animais , Coativador 3 de Receptor Nuclear , Animais , Feminino , Masculino , Camundongos , Ligantes , Camundongos Knockout , Coativador 3 de Receptor Nuclear/genética , Linfócitos T Reguladores , Tamoxifeno/farmacologiaRESUMO
Steroid receptor coactivator 3 (SRC-3) is most strongly expressed in regulatory T cells (Tregs) and B cells, suggesting that it plays an important role in the regulation of Treg function. Using an aggressive E0771 mouse breast cell line syngeneic immune-intact murine model, we observed that breast tumors were 'permanently eradicated' in a genetically engineered tamoxifen-inducible Treg-cell specific SRC-3 knockout (KO) female mouse that does not possess a systemic autoimmune pathological phenotype. A similar eradication of tumor was noted in a syngeneic model of prostate cancer. A subsequent injection of additional E0771 cancer cells into these mice showed continued resistance to tumor development without the need for tamoxifen induction to produce additional SRC-3 KO Tregs. SRC-3 KO Tregs were highly proliferative and preferentially infiltrated into breast tumors by activating the Chemokine (C-C motif) ligand (Ccl) 19/Ccl21/ Chemokine (C-C motif) Receptor (Ccr)7 signaling axis, generating antitumor immunity by enhancing the interferon-γ/C-X-C Motif Chemokine Ligand (Cxcl) 9 signaling axis to facilitate the entrance and function of effector T cells and Natural Killer cells. SRC-3 KO Tregs also show a dominant effect by blocking the immune suppressive function of WT Tregs. Importantly, a single adoptive transfer of SRC-3 KO Tregs into wild-type E0771 tumor-bearing mice can completely abolish pre-established breast tumors by generating potent antitumor immunity with a durable effect that prevents tumor reoccurrence. Therefore, treatment with SRC-3 deleted Tregs represents a novel approach to completely block tumor growth and recurrence without the autoimmune side-effects that typically accompany immune checkpoint modulators. Significance statement: Tregs are essential in restraining immune responses for immune homeostasis. SRC-3 is a pleiotropic coactivator, the second-most highly expressed transcriptional coactivator in Tregs, and a suspect in Treg function. The disruption of SRC-3 expression in Tregs leads to a 'complete lifetime eradication' of tumors in aggressive syngeneic breast cancer mouse models because deletion of SRC-3 alters the expression of a wide range of key genes involved in efferent and afferent Treg signaling. SRC-3KO Tregs confer this long-lasting protection against cancer recurrence in mice without an apparent systemic autoimmune pathological phenotype. Therefore, treatment with SRC-3 deleted Tregs could represent a novel and efficient future target for eliminating tumor growth and recurrence without the autoimmune side-effects that typically accompany immune checkpoint modulators.
RESUMO
HER2-positive (HER2+) breast cancers (BrCs) contain approximately equal numbers of ERα+HER2+ and ERα-HER2+ cases. An enduring obstacle is the unclear cell lineage-related characteristics of these BrCs. Although ERα+HER2+ BrCs could lose ERα to become ERα-HER2+ BrCs, direct evidence is missing. To investigate ERα dependencies and their implications during BrC growth and metastasis, we generated ERαCreRFP-T mice that produce an RFP-marked ERα+ mammary gland epithelial cell (MGEC) lineage. RCAS virus-mediated expression of Erbb2, a rodent Her2 homolog, first produced comparable numbers of ERα+RFP+Erbb2+ and ERα-RFP-Erbb2+ MGECs. Early hyperplasia developed mostly from ERα+RFP+Erbb2+ cells and ERα-RFP-Erbb2+ cells in these lesions were rare. The subsequently developed ductal carcinomas in situ had 64% slow-proliferating ERα+RFP+Erbb2+ cells, 15% fast-proliferating ERα-RFP+Erbb2+ cells derived from ERα+RFP+Erbb2+ cells, and 20% fast-proliferating ERα-RFP-Erbb2+ cells. The advanced tumors had mostly ERα-RFP+Erbb2+ and ERα-RFP-Erbb2+ cells and only a very small population of ERα+RFP+Erbb2+ cells. In ERα-RFP+Erbb2+ cells, GATA3 and FoxA1 decreased expression and ERα promoter regions became methylated, consistent with the loss of ERα expression. Lung metastases consisted of mostly ERα-RFP+Erbb2+ cells, a few ERα-RFP-Erbb2+ cells, and no ERα+RFP+Erbb2+ cells. The high metastatic capacity of ERα-RFP+Erbb2+ cells was associated with ERK1/2 activation. These results show that the slow-proliferating, nonmetastatic ERα+RFP+Erbb2+ cells progressively lose ERα during tumorigenesis to become fast-proliferating, highly metastatic ERα-RFP+Erbb2+ cells. The ERα-Erbb2+ BrCs with an ERα+ origin are more aggressive than those ERα-Erbb2+ BrCs with an ERα- origin, and thus, they should be distinguished and treated differently in the future.
Assuntos
Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Receptor ErbB-2/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/secundário , Linhagem Celular Tumoral , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Proliferação de Células , Transformação Celular Neoplásica , Receptor alfa de Estrogênio/metabolismo , Feminino , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Regiões Promotoras Genéticas , Receptor ErbB-2/metabolismo , Transdução de Sinais , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Current first-line treatment of patients with high-grade serous ovarian cancer (HGSOC) involves the use of cytotoxic drugs that frequently lead to recurrent tumors exhibiting increased resistance to the drugs and poor patient survival. Strong evidence is accumulating to show that HGSOC tumors and cell lines contain a subset of cells called polyploidy giant cancer cells (PGCCs) that act as stem-like, self-renewing cells. These PGCCs appear to play a key role in tumor progression by generating drug-resistant progeny produced, in part, as a consequence of utilizing a modified form of mitosis known as endoreplication. Thus, developing drugs to target PGCCs and endoreplication may be an important approach for reducing the appearance of drug-resistant progeny. In the review, we discuss newly identified regulatory factors that impact mitosis and which may be altered or repurposed during endoreplication in PGCCs. We also review recent papers showing that a single PGCC can give rise to tumors in vivo and spheroids in culture. To illustrate some of the specific features of PGCCs and factors that may impact their function and endoreplication compared to mitosis, we have included immunofluorescent images co-localizing p53 and specific mitotic regulatory, phosphoproteins in xenografts derived from commonly used HGSOC cell lines.
Assuntos
Células Gigantes/fisiologia , Neoplasias Ovarianas/genética , Poliploidia , Animais , Feminino , Humanos , Camundongos , MitoseRESUMO
Differences in the relative abundances of the progesterone receptor (PGR) isoforms PGRA and PGRB are often observed in women with reproductive tract cancers. To assess the importance of the PGR isoform ratio in the maintenance of the reproductive tract, we generated mice that overexpress PGRA or PGRB in all PGR-positive tissues. Whereas few PGRA-overexpressing mice developed reproductive tract tumors, all PGRB-overexpressing mice developed ovarian neoplasms that were derived from ovarian luteal cells. Transcriptomic analyses of the ovarian tumors from PGRB-overexpressing mice revealed enhanced AKT signaling and a gene expression signature similar to those of human ovarian and endometrial cancers. Treating PGRB-overexpressing mice with the PGR antagonist RU486 stalled tumor growth and decreased the expression of cell cycle-associated genes, indicating that tumor growth and cell proliferation were hormone dependent in addition to being isoform dependent. Analysis of the PGRB cistrome identified binding events at genes encoding proteins that are critical regulators of mitotic phase entry. This work suggests a mechanism whereby an increase in the abundance of PGRB relative to that of PGRA drives neoplasia in vivo by stimulating cell cycling.
Assuntos
Perfilação da Expressão Gênica/métodos , Hormônios/metabolismo , Neoplasias Ovarianas/genética , Receptores de Progesterona/genética , Transcriptoma/genética , Animais , Proliferação de Células/genética , Modelos Animais de Doenças , Estradiol/sangue , Estradiol/metabolismo , Feminino , Hormônios/sangue , Humanos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Neoplasias Ovarianas/metabolismo , Progesterona/sangue , Progesterona/metabolismo , Receptores de Progesterona/metabolismoRESUMO
Mouse studies support a role for endometrial early growth response 1 (EGR1) in uterine receptivity and decidualization, which are processes controlled by estrogen and progesterone. However, the importance of this transcription factor in similar cellular processes in human is unclear. Analysis of clinical samples indicate that endometrial EGR1 levels are decreased in the endometrium of women with recurrent implantation failure, suggesting that tight control of EGR1 levels are necessary for normal endometrial function. Therefore, we used siRNA-mediated knockdown of EGR1 expression in cultured human endometrial stromal cells (hESCs) to assess the functional role of EGR1 in hESC decidualization. Protein expression studies revealed that EGR1 is highly expressed in pre-decidual hESCs. However, EGR1 protein levels rapidly decrease following administration of an established deciduogenic hormone stimulus containing estradiol, medroxyprogesterone acetate, and cyclic adenosine monophosphate. Intriguingly, EGR1 knockdown in pre-decidual hESCs blocks the ability of these cells to decidualize later, indicating that EGR1 is required to transcriptionally program pre-decidual hESCs for decidualization. Support for this proposal comes from the analysis of transcriptome and cistrome datasets, which shows that EGR1 target genes are primarily involved in transcriptional regulation, cell signaling, and proliferation. Collectively, our studies provide translational support for an evolutionary conserved role for human endometrial stromal EGR1 in the early events of pregnancy establishment.
Assuntos
Decídua/citologia , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Endométrio/citologia , Células Estromais/citologia , Animais , Células Cultivadas , Decídua/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Implantação do Embrião , Endométrio/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Gravidez , Células Estromais/metabolismo , Ativação TranscricionalRESUMO
Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, ß-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.
Assuntos
Implantação do Embrião/genética , Implantação do Embrião/fisiologia , Endométrio/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Animais , Núcleo Celular/metabolismo , Polaridade Celular/genética , Polaridade Celular/fisiologia , Decídua/fisiologia , Endométrio/citologia , Feminino , Proteína Forkhead Box O1/deficiência , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Gravidez , Receptores de Progesterona/deficiência , Transdução de SinaisRESUMO
Mammalian pregnancy depends on the ability of the uterus to support embryo implantation. Previous studies reveal the Sox17 gene as a downstream target of the Pgr-Gata2-dependent transcription network that directs genomic actions in the uterine endometrium receptive for embryo implantation. Here, we report that ablating Sox17 in the uterine epithelium impairs leukemia inhibitory factor (LIF) and Indian hedgehog homolog (IHH) signaling, leading to failure of embryo implantation. In vivo deletion of the SOX17-binding region 19 kb upstream of the Ihh locus by CRISPR-Cas technology reduces Ihh expression specifically in the uterus and alters proper endometrial epithelial-stromal interactions, thereby impairing pregnancy. This SOX17-binding interval is also bound by GATA2, FOXA2, and PGR. This cluster of transcription factor binding is common in 737 uterine genes and may represent a key regulatory element essential for uterine epithelial gene expression.
Assuntos
Proteínas HMGB/metabolismo , Fatores de Transcrição SOXF/metabolismo , Útero/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Endométrio/metabolismo , Feminino , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Proteínas HMGB/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Camundongos , Fatores de Transcrição SOXF/genética , Transcriptoma/genéticaRESUMO
Establishment of a successful pregnancy requires not only implantation of a healthy embryo into a receptive uterus but also progesterone receptor (PGR)-dependent transformation of endometrial stromal cells (ESCs) into specialized decidual cells. Decidual cells support the developing embryo and are critical for placentation. We have previously shown that a known transcriptional coregulator of the PGR, steroid receptor coactivator-2 (SRC-2), is a critical driver of endometrial decidualization in both human and mouse endometrium. However, the full spectrum of genes transcriptionally controlled by SRC-2 in decidualizing ESCs has not been identified. Therefore, using an RNA- and chromatin immunoprecipitation-sequencing approach, we have identified the transcriptome of decidualizing human ESCs (hESCs) that requires SRC-2. We revealed that the majority of hESC genes regulated by SRC-2 are associated with decidualization. Over 50% of SRC-2-regulated genes are also controlled by the PGR. While ontology analysis showed that SRC-2-dependent genes are functionally linked to signaling processes known to underpin hESC decidualization, cell membrane processes were significantly enriched in this analysis. Follow-up studies showed that retinoid signaling is dependent on SRC-2 during hESC decidualization. Specifically, SRC-2 is required for full induction of the retinol transporter, stimulated by retinoic acid 6 (STRA6), which is essential for hESC decidualization. Together our findings show that a critical subset of genes transcriptionally reprogramed by PGR during hESC decidualization requires SRC-2. Among the multiple genes, pathways and networks that are dependent on SRC-2 during hESC decidualization, first-line analysis supports a critical role for this coregulator in maintaining retinoid signaling during progesterone-driven decidualization.
Assuntos
Endométrio/fisiologia , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Coativador 2 de Receptor Nuclear/fisiologia , Transcriptoma , Células Cultivadas , Feminino , Humanos , Receptores de Progesterona/metabolismo , Análise de Sequência de RNARESUMO
Steroid receptor coactivator-2 (SRC-2) is a transcriptional coregulator that modulates the activity of many transcription factors. Levels of SRC-2 are elevated in endometrial biopsies from polycystic ovary syndrome patients, a population predisposed to endometrial cancer (EC). Increased expression of SRC-2 is also detected in neoplastic endometrium suggesting a causal link between elevated SRC-2 expression and the emergence of endometrial disorders that can lead to cancer. Here, we reveal that SRC-2 knockdown reduces EC cell proliferation and anchorage-independence. Additionally, SRC-2 is required to maintain cellular glycolytic capacity and oxidative phosphorylation, processes essential for EC cell proliferation. Importantly, SRC-2 is critical for the normal performance of the pentose phosphate pathway (PPP). Perturbation of the PPP due to loss of SRC-2 expression may result from the depletion of ribose-5-P isomerase (RPIA), a key enzyme of the PPP. As with SRC-2, RPIA knockdown reduces EC cell proliferation, which is accompanied by a decrease in glycolytic capacity and oxidative phosphorylation. Glucose metabolite tracking experiments confirmed that knockdown of SRC-2 and RPIA downregulates the metabolic rate of both glycolysis and the PPP, highlighting a novel regulatory cross-talk between glycolysis and the PPP modulated by SRC-2.
Assuntos
Aldose-Cetose Isomerases/genética , Endométrio/metabolismo , Regulação Neoplásica da Expressão Gênica , Coativador 2 de Receptor Nuclear/genética , Via de Pentose Fosfato/genética , Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/metabolismo , Isótopos de Carbono , Linhagem Celular Tumoral , Proliferação de Células , Endométrio/patologia , Feminino , Glicólise/genética , Humanos , Metaboloma/genética , Coativador 2 de Receptor Nuclear/antagonistas & inibidores , Coativador 2 de Receptor Nuclear/metabolismo , Fosforilação Oxidativa , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
RNA polymerase (pol) III transcribes a variety of small untranslated RNAs involved in transcription, RNA processing, and translation. RNA pol III and its components are altered in various human developmental disorders, yet their roles in cell fate determination and development are poorly understood. Here we demonstrate that Maf1, a transcriptional repressor, promotes induction of mouse embryonic stem cells (mESCs) into mesoderm. Reduced Maf1 expression in mESCs and preadipocytes impairs adipogenesis, while ectopic Maf1 expression in Maf1-deficient cells enhances differentiation. RNA pol III repression by chemical inhibition or knockdown of Brf1 promotes adipogenesis. Altered RNA pol III-dependent transcription produces select changes in mRNAs with a significant enrichment of adipogenic gene signatures. Furthermore, RNA pol III-mediated transcription positively regulates long non-coding RNA H19 and Wnt6 expression, established adipogenesis inhibitors. Together, these studies reveal an important and unexpected function for RNA pol III-mediated transcription and Maf1 in mesoderm induction and adipocyte differentiation.
Assuntos
Adipócitos/metabolismo , Adipogenia/genética , RNA Polimerase III/genética , Proteínas Repressoras/genética , Transcrição Gênica , Adipócitos/citologia , Animais , Fator 1 de Resposta a Butirato , Diferenciação Celular , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Mesoderma/citologia , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Camundongos Nus , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Polimerase III/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama/genética , Cromatina/metabolismo , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica , Coativador 3 de Receptor Nuclear/metabolismo , Transcrição Gênica , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cromatina/genética , Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Coativador 3 de Receptor Nuclear/genética , Regiões Promotoras Genéticas , Ligação Proteica , Células Tumorais CultivadasRESUMO
Infertility and early embryo miscarriage is linked to inadequate endometrial decidualization. Although transcriptional reprogramming is known to drive decidualization in response to progesterone, the key signaling effectors that directly mediate this hormone response are not fully known. This knowledge gap is clinically significant because identifying the early signals that directly mediate progesterone-driven decidualization will address some of the current limitations in diagnosing and therapeutically treating patients at most risk for early pregnancy loss. We recently revealed that the promyelocytic leukemia zinc finger (PLZF) is a direct target of the progesterone receptor and is essential for decidualization of human endometrial stromal cells (hESCs). The purpose of this current work was to identify the genome-wide transcriptional program that is controlled by PLZF during hESC decidualization using an established in vitro hESC culture model, siRNA-mediated knockdown methods, and RNA-sequencing technology followed by bioinformatic analysis and validation. We discovered that PLZF is critical in the regulation of genes that are involved in cellular processes that are essential for the archetypal morphological and functional changes that occur when hESCs transform into epithelioid decidual cells such as proliferation and cell motility. We predict that the transcriptome datasets identified in this study will not only contribute to a broader understanding of PLZF-dependent endometrial decidualization at the molecular level but may advance the development of more effective molecular diagnostics and therapeutics for the clinical management of female infertility and subfertility that is based on a dysfunctional endometrium.
Assuntos
Decídua/fisiologia , Endométrio/citologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Biologia Computacional , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Interferência de RNA , Células Estromais/citologia , Células Estromais/metabolismo , TranscriptomaRESUMO
The precise timing of progesterone signaling through its cognate receptor, the progesterone receptor (PGR), is critical for the establishment and maintenance of pregnancy. Loss of PGR expression in the murine uterine epithelium during the preimplantation period is a marker for uterine receptivity and embryo attachment. We hypothesized that the decrease in progesterone receptor A (PGRA) expression is necessary for successful embryo implantation. To test this hypothesis, a mouse model constitutively expressing PGRA (mPgrALsL/+) was generated. Expression of PGRA in all uterine compartments (Pgrcre) or uterine epithelium (Wnt7acre) resulted in infertility with defects in embryo attachment and stromal decidualization. Expression of critical PGRA target genes, indian hedgehog, and amphiregulin (Areg), was maintained through the window of receptivity while the estrogen receptor target gene, the leukemia inhibitory factor (Lif), a key regulator of embryo receptivity, was decreased. Transcriptomic and cistromic analyses of the mouse uterus at day 4.5 of pregnancy identified an altered group of genes regulating molecular transport in the control of fluid and ion levels within the uterine interstitial space. Additionally, LIF and its cognate receptor, the leukemia inhibitory factor receptor (LIFR), exhibited PGR-binding events in regions upstream of the transcriptional start sites, suggesting PGRA is inhibiting transcription at these loci. Therefore, downregulation of the PGRA isoform at the window of receptivity is necessary for the attenuation of hedgehog signaling, transcriptional activation of LIF signaling, and modulation of solutes and fluid, producing a receptive environment for the attaching embryo.
Assuntos
Implantação do Embrião , Endométrio , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Alelos , Animais , Clonagem Molecular , Regulação para Baixo , Feminino , Regulação da Expressão Gênica/fisiologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Masculino , Camundongos Transgênicos , Receptores de OSM-LIF/genética , Receptores de OSM-LIF/metabolismo , Receptores de Progesterona/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
During neonatal testis development, centrally located gonocytes migrate to basement membrane of the seminiferous cords, where physical contact with a niche established by Sertoli cells is essential for transition of gonocytes into spermatogonial stem cells (SSCs). To provide structural support and signaling stimuli for the gonocyte-to-SSC transition that occurs at a specific location during a finite phase, temporal-spatial establishment of the niche is critical. To date, the factors that guide Sertoli cells to establish the initial stem cell niche remain largely unknown. Using the Sertoli cell-specific Arid4b knockout (Arid4bSCKO) mice, we demonstrated that ablation of AT-rich interaction domain 4B (ARID4B) resulted in abnormal detachment of Sertoli cells from the basement membrane of seminiferous cords during the gonocyte-to-SSC transition phase, suggesting failure to establish a niche for the SSC formation. Without support by a niche environment, gonocytes showed disarranged cell distribution in the Arid4bSCKO testes and underwent apoptosis. The commitment of gonocytes to differentiate into the spermatogonial lineage was broken and the capability of SSCs to self-renew and differentiate was also impaired. Gene expression profiling revealed the molecular mechanisms responsible for the phenotypic changes in the Arid4bSCKO testes, by identifying genes important for stem cell niche function as downstream effectors of ARID4B, including genes that encode gap junction protein alpha-1, KIT ligand, anti-Müllerian hormone, Glial cell-line derived neurotrophic factor, inhibin alpha, inhibin beta, and cytochrome P450 family 26 subfamily b polypeptide 1. Our results identified ARID4B as a master regulator of a signaling network that governs the establishment of a niche during the critical gonocyte-to-SSC transition phase to control the fate of gonocytes and SSCs. Stem Cells 2017;35:1554-1565.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Redes Reguladoras de Genes , Espermatogônias/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Diferenciação Celular , Linhagem da Célula , Autorrenovação Celular , Proteínas de Ligação a DNA/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica no Desenvolvimento , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Testículo/embriologia , Testículo/metabolismo , Fatores de TempoRESUMO
Altered progesterone responsiveness leads to female infertility and cancer, but underlying mechanisms remain unclear. Mice with uterine-specific ablation of GATA binding protein 2 (Gata2) are infertile, showing failures in embryo implantation, endometrial decidualization, and uninhibited estrogen signaling. Gata2 deficiency results in reduced progesterone receptor (PGR) expression and attenuated progesterone signaling, as evidenced by genome-wide expression profiling and chromatin immunoprecipitation. GATA2 not only occupies at and promotes expression of the Pgr gene but also regulates downstream progesterone responsive genes in conjunction with the PGR. Additionally, Gata2 knockout uteri exhibit abnormal luminal epithelia with ectopic TRP63 expressing squamous cells and a cancer-related molecular profile in a progesterone-independent manner. Lastly, we found a conserved GATA2-PGR regulatory network in both human and mice based on gene signature and path analyses using gene expression profiles of human endometrial tissues. In conclusion, uterine Gata2 regulates a key regulatory network of gene expression for progesterone signaling at the early pregnancy stage.
Assuntos
Endométrio/metabolismo , Redes Reguladoras de Genes/genética , Progesterona/metabolismo , Animais , Sequência de Bases , Sequência Conservada/genética , Implantação do Embrião , Feminino , Fator de Transcrição GATA2/metabolismo , Humanos , Camundongos , Fosfoproteínas/metabolismo , Gravidez , Progesterona/sangue , Ligação Proteica/genética , Receptores de Progesterona/metabolismo , Transdução de Sinais/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: TMPRSS2-ERG fusion occurs in about half of prostate cancers and results in over-expression of the oncogenic ERG protein in the prostate. The mechanism by which ERG contributes to prostate cancer initiation and progression remains largely unknown. Because ERG is a transcriptional activator, we reasoned that the target genes regulated by ERG could contribute to prostate cancer development. METHODS: In a search for ERG target genes, we took advantage of published datasets from the MSKCC Prostate Oncogene Project, in which a comprehensive analysis was applied to define transcriptomes in 150 prostate tumors. We retrieved the mRNA expression dataset, split them based on ERG expression, and identified genes whose expression levels are associated with ERG mRNA levels. RESULTS: mRNA expression levels of 21 genes were found to be significantly increased, while for one gene it was decreased in ERG-positive prostate tumors. Among them, the expression of TDRD1 was the most significantly increased in ERG-positive tumors. Among 131 primary prostate tumors which were primarily from European American patients, TDRD1 is over-expressed in 68% of samples, while ERG is overexpressed in 48% of samples, suggesting an additional ERG-independent mechanism of TDRD1 overexpression. In African American prostate tumors, TDRD1 mRNA is expressed in 44%, while ERG is expressed in 24% of samples. In normal tissues, TDRD1 mRNA is exclusively expressed in germ cells and its protein is also known as cancer/testis antigen 41.1 (CT41.1). We generated a mouse monoclonal antibody that recognizes human TDRD1 protein with high specificity and sensitivity. By Western blot analysis and immunohistochemistry (IHC) staining, we demonstrate that TDRD1 protein is expressed in the majority of human prostate tumors, but not in normal prostate tissue. Finally, TDRD1 is not induced in the prostate of ERG overexpression transgenic mice, suggesting that such model does not fully recapitulate the TMPRSS2/ERG fusion-dependent human prostate cancer development. CONCLUSIONS: Our results suggest TDRD1 as a novel prostate cancer biomarker. As an ERG target gene, TDRD1 might play an important role in human prostate cancer development, and as a cancer/testis antigen, TDRD1 might have long-term potential to be a therapeutic target for prostate cancer immunotherapy. Prostate 76:1271-1284, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Marcação de Genes/métodos , Células Germinativas , Neoplasias da Próstata/genética , Animais , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Células Germinativas/metabolismo , Células Germinativas/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Regulador Transcricional ERG/biossíntese , Regulador Transcricional ERG/genéticaRESUMO
The TMPRSS2-ERG fusion occurs in approximately 50% of prostate cancer (PCa), resulting in expression of the oncogenic ERG in the prostate. Because ERG is a transcriptional activator, we hypothesized that ERG-regulated genes contribute to PCa development. Since microRNA (miRNA) has crucial functions in cancer, we searched for miRNAs regulated by ERG in PCas. We mined published datasets based on the MSKCC Prostate Oncogene Project, in which a comprehensive analysis defined the miRNA transcriptomes in 113 PCas. We retrieved the miRNA expression datasets, and identified miRNAs differentially expressed between ERG-positive and ERG-negative samples. Out of 369 miRNAs, miR-200a, -200b, -429 and -205 are the only miRNAs significantly increased in ERG-positive tumors. Strikingly, miR-200a, -200b and -429 are transcribed as a single polycistronic transcript, suggesting they are regulated at the transcriptional level. With ChIP-qPCR and in vitro binding assay, we identified two functional ETS motifs in the miR-200b/a/429 gene promoter. Knockdown of ERG in PCa cells reduced expression of these three miRNAs. In agreement with the well-established tumor suppressor function, overexpression of the miR-200b/a/429 gene inhibited PCa cell growth and invasion. In summary, our study reveals that miR-200b/a/429 is an ERG target gene, which implicates an important role in TMPRSS2/ERG-dependent PCa development. Although induction of the tumor suppressive miR-200b subfamily by oncogenic ERG appears to be counterintuitive, it is consistent with the observation that the vast majority of primary prostate cancers are slow-growing and indolent.
Assuntos
MicroRNAs/genética , Neoplasias da Próstata/genética , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias da Próstata/patologia , Regulador Transcricional ERG/genética , TransfecçãoRESUMO
Progesterone, via the progesterone receptor (PGR), is essential for endometrial stromal cell decidualization, a cellular transformation event in which stromal fibroblasts differentiate into decidual cells. Uterine decidualization supports embryo implantation and placentation as well as subsequent events, which together ensure a successful pregnancy. Accordingly, impaired decidualization results not only in implantation failure or early fetal miscarriage, but also may lead to potential adverse outcomes in all three pregnancy trimesters. Transcriptional reprogramming on a genome-wide scale underlies progesterone dependent decidualization of the human endometrial stromal cell (hESC). However, identification of the functionally essential signals encoded by these global transcriptional changes remains incomplete. Importantly, this knowledge-gap undercuts future efforts to improve diagnosis and treatment of implantation failure based on a dysfunctional endometrium. By integrating genome-wide datasets derived from decidualization of hESCs in culture, we reveal that the promyelocytic leukemia zinc finger (PLZF) transcription factor is rapidly induced by progesterone and that this induction is indispensable for progesterone-dependent decidualization. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) identified at least ten progesterone response elements within the PLZF gene, indicating that PLZF may act as a direct target of PGR signaling. The spatiotemporal expression profile for PLZF in both the human and mouse endometrium offers further support for stromal PLZF as a mediator of the progesterone decidual signal. To identify functional targets of PLZF, integration of PLZF ChIP-Seq and RNA Pol II RNA-Seq datasets revealed that the early growth response 1 (EGR1) transcription factor is a PLZF target for which its level of expression must be reduced to enable progesterone dependent hESC decidualization. Apart from furnishing essential insights into the molecular mechanisms by which progesterone drives hESC decidualization, our findings provide a new conceptual framework that could lead to new avenues for diagnosis and/or treatment of adverse reproductive outcomes associated with a dysfunctional uterus.