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1.
Nat Commun ; 14(1): 2439, 2023 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-37117180

RESUMO

Cancer cells undergo transcriptional reprogramming to drive tumor progression and metastasis. Using cancer cell lines and patient-derived tumor organoids, we demonstrate that loss of the negative elongation factor (NELF) complex inhibits breast cancer development through downregulating epithelial-mesenchymal transition (EMT) and stemness-associated genes. Quantitative multiplexed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (qPLEX-RIME) further reveals a significant rewiring of NELF-E-associated chromatin partners as a function of EMT and a co-option of NELF-E with the key EMT transcription factor SLUG. Accordingly, loss of NELF-E leads to impaired SLUG binding on chromatin. Through integrative transcriptomic and genomic analyses, we identify the histone acetyltransferase, KAT2B, as a key functional target of NELF-E-SLUG. Genetic and pharmacological inactivation of KAT2B ameliorate the expression of EMT markers, phenocopying NELF ablation. Elevated expression of NELF-E and KAT2B is associated with poorer prognosis in breast cancer patients, highlighting the clinical relevance of our findings. Taken together, we uncover a crucial role of the NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis.


Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/patologia , Carcinogênese/genética , Linhagem Celular Tumoral , Cromatina , Epigênese Genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição de p300-CBP/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição/metabolismo
2.
Elife ; 92020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32706336

RESUMO

There is strong evidence that the pro-inflammatory microenvironment during post-partum mammary involution promotes parity-associated breast cancer. Estrogen exposure during mammary involution drives tumor growth through neutrophils' activity. However, how estrogen and neutrophils influence mammary involution are unknown. Combined analysis of transcriptomic, protein, and immunohistochemical data in BALB/c mice showed that estrogen promotes involution by exacerbating inflammation, cell death and adipocytes repopulation. Remarkably, 88% of estrogen-regulated genes in mammary tissue were mediated through neutrophils, which were recruited through estrogen-induced CXCR2 signalling in an autocrine fashion. While neutrophils mediate estrogen-induced inflammation and adipocytes repopulation, estrogen-induced mammary cell death was via lysosome-mediated programmed cell death through upregulation of cathepsin B, Tnf and Bid in a neutrophil-independent manner. Notably, these multifaceted effects of estrogen are mostly mediated by ERα and unique to the phase of mammary involution. These findings are important for the development of intervention strategies for parity-associated breast cancer.


Assuntos
Neoplasias da Mama/fisiopatologia , Estrogênios/metabolismo , Regulação Neoplásica da Expressão Gênica , Glândulas Mamárias Animais/efeitos dos fármacos , Neoplasias Mamárias Animais/fisiopatologia , Neutrófilos/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Glândulas Mamárias Animais/fisiologia , Glândulas Mamárias Animais/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Paridade , Período Pós-Parto , Regulação para Cima
4.
ACS Med Chem Lett ; 10(6): 978-984, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31223458

RESUMO

SMYD3 is a histone methyltransferase that regulates gene transcription, and its overexpression is associated with multiple human cancers. A novel class of tetrahydroacridine compounds which inhibit SMYD3 through a covalent mechanism of action is identified. Optimization of these irreversible inhibitors resulted in the discovery of 4-chloroquinolines, a new class of covalent warheads. Tool compound 29 exhibits high potency by inhibiting SMYD3's enzymatic activity and showing antiproliferative activity against HepG2 in 3D cell culture. Our findings suggest that covalent inhibition of SMYD3 may have an impact on SMYD3 biology by affecting expression levels, and this warrants further exploration.

5.
Biochim Biophys Acta Gene Regul Mech ; 1862(4): 522-533, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30716532

RESUMO

The activation functions AF1 and AF2 of nuclear receptors mediate the recruitment of coregulators in gene regulation. AF1 is mapped to the highly variable and intrinsically unstructured N terminal domain and AF2 lies in the conserved ligand binding domain. The unstructured nature of AF1 offers structural plasticity and hence functional versatility in gene regulation. However, little is known about the key functional residues of AF1 that mediates its interaction with coregulators. This study focuses on the progesterone receptor (PR) and reports the identification of K464, K481 and R492 (KKR) as the key functional residues of PR AF1. The KKR are monomethylated and function cooperatively. The combined mutations of KKR to QQQ render PR isoform B (PRB) hyperactive, whereas KKR to FFF mutations abolishes as much as 80% of PR activity. Furthermore, the hyperactive QQQ mutation rescues the loss of PR activity due to E911A mutation in AF2. The study also finds that the magnitudes of the mutational effect differ in different cell types as a result of differential effects on the functional interaction with coregulators. Furthermore, KKR provides the interface for AF1 to physically interact with p300 and SRC-1, and with AF2 at E911. Intriguingly, the inactive FFF mutant interacts strikingly stronger with both SRC-1 and AF2 than wt PRB. We propose a tripartite model to describe the dynamic interactions between AF1, AF2 and SRC-1 with KKR of AF1 and E911 of AF2 as the interface. An overly stable interaction would hamper the dynamics of disassembly of the receptor complex.


Assuntos
Aminoácidos/química , Receptores de Progesterona/química , Receptores de Progesterona/metabolismo , Linhagem Celular Tumoral , Humanos , Ligantes , Metilação , Mutação , NF-kappa B/metabolismo , Coativador 1 de Receptor Nuclear/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores de Progesterona/genética , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica , Fatores de Transcrição de p300-CBP/metabolismo
6.
Sci Rep ; 7: 46485, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28429725

RESUMO

Epidemiological studies have indicated increased risk for breast cancer within 10 years of childbirth. Acute inflammation during mammary involution has been suggested to promote this parity-associated breast cancer. We report here that estrogen exacerbates mammary inflammation during involution. Microarray analysis shows that estrogen induces an extensive proinflammatory gene signature in the involuting mammary tissue. This is associated with estrogen-induced neutrophil infiltration. Furthermore, estrogen induces the expression of protumoral cytokines/chemokines, COX-2 and tissue-remodeling enzymes in isolated mammary neutrophils and systemic neutrophil depletion abolished estrogen-induced expression of these genes in mammary tissue. More interestingly, neutrophil depletion diminished estrogen-induced growth of ERα-negative mammary tumor 4T1 in Balb/c mice. These findings highlight a novel aspect of estrogen action that reprograms the activity of neutrophils to create a pro-tumoral microenvironment during mammary involution. This effect on the microenvironment would conceivably aggravate its known neoplastic effect on mammary epithelial cells.


Assuntos
Reprogramação Celular , Estrogênios/metabolismo , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Neutrófilos/metabolismo , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/biossíntese , Neutrófilos/patologia
7.
Sci Rep ; 6: 37568, 2016 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-27869229

RESUMO

Tetratricopeptide repeat domain 9A (TTC9A) expression is abundantly expressed in the brain. Previous studies in TTC9A knockout (TTC9A-/-) mice have indicated that TTC9A negatively regulates the action of estrogen. In this study we investigated the role of TTC9A on anxiety-like behavior through its functional interaction with estrogen using the TTC9A-/- mice model. A battery of tests on anxiety-related behaviors was conducted. Our results demonstrated that TTC9A-/- mice exhibited an increase in anxiety-like behaviors compared to the wild type TTC9A+/+ mice. This difference was abolished after ovariectomy, and administration of 17-ß-estradiol benzoate (EB) restored this escalated anxiety-like behavior in TTC9A-/- mice. Since serotonin is well-known to be the key neuromodulator involved in anxiety behaviors, the mRNA levels of tryptophan hydroxylase (TPH) 1, TPH2 (both are involved in serotonin synthesis), and serotonin transporter (5-HTT) were measured in the ventromedial prefrontal cortex (vmPFC) and dorsal raphe nucleus (DRN). Interestingly, the heightened anxiety in TTC9A-/- mice under EB influence is consistent with a greater induction of TPH 2, and 5-HTT by EB in DRN that play key roles in emotion regulation. In conclusion, our data indicate that TTC9A modulates the anxiety-related behaviors through modulation of estrogen action on the serotonergic system in the DRN.


Assuntos
Ansiedade/metabolismo , Comportamento Animal , Proteínas dos Microfilamentos/metabolismo , Animais , Ansiedade/fisiopatologia , Núcleo Dorsal da Rafe/metabolismo , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Fenótipo , Filosofia , Córtex Pré-Frontal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serotonina/genética , Serotonina/metabolismo , Triptofano Hidroxilase/metabolismo
8.
Biochim Biophys Acta ; 1843(9): 2067-78, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24742914

RESUMO

Recent studies reported that protein arginine methyltransferase 6 (PRMT6) enhances estrogen-induced activity of estrogen receptor α (ERα) and dysfunction of PRMT6 is associated with overall better survival for ERα-positive breast cancer patients. However, it is unclear how PRMT6 promotes ERα activity. Here we report that PRMT6 specifically interacts with ERα at its ligand-binding domain. PRMT6 also methylates ERα both in vitro and in vivo. In addition to enhancing estrogen-induced ERα activity, PRMT6 over-expression up-regulates estrogen-independent activity of ERα and PRMT6 gene silencing in MCF7 cells inhibits ligand-independent ERα activation. More interestingly, the effect of PRMT6 on the ligand-independent ERα activity does not require its methyltransferase activity. Instead, PRMT6 competes with Hsp90 for ERα binding: PRMT6 and Hsp90 bindings to ERα are mutually exclusive and PRMT6 over-expression reduces ERα interaction with Hsp90. In conclusion, PRMT6 requires its methyltransferase activity to enhance ERα's ligand-induced activity, but its effect on ligand-independent activity is likely mediated through competing with Hsp90 for binding to the C-terminal domain of ERα. PRMT6-ERα interaction would prevent ERα-Hsp90 association. Since Hsp90 and associated chaperones serve to maintain ERα conformation for ligand-binding yet functionally inactive, inhibition of ERα-Hsp90 interaction would relieve ERα from the constraint of chaperone complex.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arginina/metabolismo , Ligação Competitiva , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Núcleo Celular/metabolismo , Proliferação de Células , Receptor alfa de Estrogênio/química , Feminino , Inativação Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Ligantes , Células MCF-7 , Metilação , Dados de Sequência Molecular , Ligação Proteica , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Regulação para Cima , Quinases da Família src/metabolismo
9.
J Biol Chem ; 289(9): 5704-22, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24415758

RESUMO

Progesterone receptor (PR) exists in two isoforms, PRA and PRB, and both contain activation functions AF-1 and AF-2. It is believed that AF-1 is primarily responsible for the ligand-independent activity, whereas AF-2 mediates ligand-dependent PR activation. Although more than a dozen post-translational modifications of PR have been reported, no post-translational modification on AF-1 or AF-2 has been reported. Using LC-MS/MS-based proteomic analysis, this study revealed AF-1 monomethylation at Lys-464. Mutational analysis revealed the remarkable importance of Lys-464 in regulating PR activity. Single point mutation K464Q or K464A led to ligand-independent PR gel upshift similar to the ligand-induced gel upshift. This upshift was associated with increases in both ligand-dependent and ligand-independent PR phosphorylation and PR activity due to the hyperactivation of AF-1. In contrast, mutation of Lys-464 to the bulkier phenylalanine to mimic the effect of methylation caused a drastic decrease in PR activity. Importantly, PR-K464Q also showed heightened ligand sensitivity, and this was associated with increases in its functional interaction with transcription co-regulators NCoR1 and SRC-1. These results suggest that monomethylation of PR at Lys-464 probably has a repressive effect on AF-1 activity and ligand sensitivity.


Assuntos
Receptores de Progesterona/metabolismo , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Humanos , Ligantes , Lisina , Metilação , Mutação de Sentido Incorreto , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Fosforilação/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Progesterona/genética
10.
J Biol Chem ; 289(4): 2180-94, 2014 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-24302725

RESUMO

The identification of lysine acetylation of steroid hormone receptors has previously been based on the presence of consensus motif (K/R)XKK. This study reports the discovery by mass spectrometry of a novel progesterone receptor acetylation site at Lys-183 that is not in the consensus motif. In vivo acetylation and mutagenesis experiments revealed that Lys-183 is a primary site of progesterone receptor (PR) acetylation. Lys-183 acetylation is enhanced by p300 overexpression and abrogated by p300 gene silencing, suggesting that p300 is the major acetyltransferase for Lys-183 acetylation. Furthermore, p300-mediated Lys-183 acetylation is associated with heightened PR activity. Accordingly, the acetylation-mimicking mutant PRB-K183Q exhibited accelerated DNA binding kinetics and greater activity compared with the wild-type PRB on genes containing progesterone response element. In contrast, Lys-183 acetylation had no influence on PR tethering effect on the nuclear factor κ-light chain enhancer of activated B cells (NFκB). Additionally, increases of Lys-183 acetylation by p300 overexpression or inhibition of deacetylation resulted in increases of Ser-294 phosphorylation levels. In conclusion, PR acetylation at Lys-183 by p300 potentiates PR activity through accelerated binding of its direct target genes without affecting PR tethering on other transcription factors. The effect may be mediated by enhancing Ser-294 phosphorylation.


Assuntos
Receptores de Progesterona/metabolismo , Ativação Transcricional/fisiologia , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Mutação de Sentido Incorreto , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação/fisiologia , Receptores de Progesterona/genética , Fatores de Transcrição de p300-CBP/genética
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