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In vitro, ACE2 translocates to the nucleus to induce SARS-CoV-2 replication. Here, using digital spatial profiling of lung tissues from SARS-CoV-2-infected golden Syrian hamsters, we show that a specific and selective peptide inhibitor of nuclear ACE2 (NACE2i) inhibits viral replication two days after SARS-CoV-2 infection. Moreover, the peptide also prevents inflammation and macrophage infiltration, and increases NK cell infiltration in bronchioles. NACE2i treatment increases the levels of the active histone mark, H3K27ac, restores host translation in infected hamster bronchiolar cells, and leads to an enrichment in methylated ACE2 in hamster bronchioles and lung macrophages, a signature associated with virus protection. In addition, ACE2 methylation is increased in myeloid cells from vaccinated patients and associated with reduced SARS-CoV-2 spike protein expression in monocytes from individuals who have recovered from infection. This protective epigenetic scarring of ACE2 is associated with a reduced latent viral reservoir in monocytes/macrophages and enhanced immune protection against SARS-CoV-2. Nuclear ACE2 may represent a therapeutic target independent of the variant and strain of viruses that use the ACE2 receptor for host cell entry.
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COVID-19 , SARS-CoV-2 , Cricetinae , Animales , Humanos , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/metabolismo , Pulmón/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Péptidos/metabolismo , Epigénesis GenéticaRESUMEN
Objective: Lysine-Specific Demethylase-1 (LSD1) is overexpressed in breast cancer cells and facilitate mesenchymal properties which may contribute to therapeutic resistance and cancer progression. The purpose of this study was to investigate the safety of combination, nab-paclitaxel and phenelzine, an irreversible LSD1 inhibitor in patients with metastatic breast cancer (mBC). Methods: Eligible patients with mBC were treated with nab-paclitaxel (100mg/m2) weekly for 3 weeks with one week break in a 28-day cycle. Dose escalation of phenelzine followed the Cumulative Cohort Design and phenelzine treatment commenced from day 2 of first cycle. Eleven patients were screened, and eligible patients were enrolled in cohorts with the dose of phenelzine ranging from 45mg to 90mg. Results: The Optimum Biological Dose was established at 60mg of phenelzine daily in combination with nab-paclitaxel and considered as the recommended phase 2 dose. Most (95%) of adverse events were grade 1 or 2 with two grade 3 events being diarrhea and neutropenia at 45mg and 60mg phenelzine respectively, with no unexpected toxicity/deaths. Commonly reported toxicities were fatigue (n=4,50%), dizziness (n=6,75%), neutropenia (n=3,37.5%), peripheral neuropathy (n=3,37.5%), diarrhea (n=2,25%), and hallucination (n=2,25%). After a median follow up of 113 weeks, all patients showed disease progression on trial with 4 patients being alive at the time of data cut off, including one patient with triple negative breast cancer. Median progression-free survival was 34 weeks. Significant inhibition of LSD1 and suppression of mesenchymal markers in circulating tumor cells were noted. Conclusion: Phenelzine in combination with nab-paclitaxel was well tolerated, without any unexpected toxicities in patients with mBC and demonstrated evidence of antitumor activity. For the first time, this proof-of-concept study showed in-vivo inhibition of LSD1 suppressed mesenchymal markers, which are known to facilitate generation of cancer stem cells with metastatic potential. Clinical Trial Registration: ClinicalTrials.Gov NCT03505528, UTN of U1111-1197-5518.
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Protein kinase C (PKC)-θ is a serine/threonine kinase with both cytoplasmic and nuclear functions. Nuclear chromatin-associated PKC-θ (nPKC-θ) is increasingly recognized to be pathogenic in cancer, whereas its cytoplasmic signaling is restricted to normal T-cell function. Here we show that nPKC-θ is enriched in circulating tumor cells (CTCs) in patients with triple-negative breast cancer (TNBC) brain metastases and immunotherapy-resistant metastatic melanoma and is associated with poor survival in immunotherapy-resistant disease. To target nPKC-θ, we designed a novel PKC-θ peptide inhibitor (nPKC-θi2) that selectively inhibits nPKC-θ nuclear translocation but not PKC-θ signaling in healthy T cells. Targeting nPKC-θ reduced mesenchymal cancer stem cell signatures in immunotherapy-resistant CTCs and TNBC xenografts. PKC-θ was also enriched in the nuclei of CD8+ T cells isolated from stage IV immunotherapy-resistant metastatic cancer patients. We show for the first time that nPKC-θ complexes with ZEB1, a key repressive transcription factor in epithelial-to-mesenchymal transition (EMT), in immunotherapy-resistant dysfunctional PD1+/CD8+ T cells. nPKC-θi2 inhibited the ZEB1/PKC-θ repressive complex to induce cytokine production in CD8+ T cells isolated from patients with immunotherapy-resistant disease. These data establish for the first time that nPKC-θ mediates immunotherapy resistance via its activity in CTCs and dysfunctional CD8+ T cells. Disrupting nPKC-θ but retaining its cytoplasmic function may offer a means to target metastases in combination with chemotherapy or immunotherapy.
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The mechanisms by which sex is determined, and how a sexual phenotype is stably maintained during adulthood, have been the focus of vigorous scientific inquiry. Resources common to the biomedical field (automated staining and imaging platforms) were leveraged to provide the first immunofluorescent data for a reptile species with temperature induced sex reversal. Two four-plex immunofluorescent panels were explored across three sex classes (sex reversed ZZf females, normal ZWf females, and normal ZZm males). One panel was stained for chromatin remodeling genes JARID2 and KDM6B, and methylation marks H3K27me3, and H3K4me3 (Jumonji Panel). The other CaRe panel stained for environmental response genes CIRBP and RelA, and H3K27me3 and H3K4me3. Our study characterized tissue specific expression and cellular localization patterns of these proteins and histone marks, providing new insights to the molecular characteristics of adult gonads in a dragon lizard Pogona vitticeps. The confirmation that mammalian antibodies cross react in P. vitticeps paves the way for experiments that can take advantage of this new immunohistochemical resource to gain a new understanding of the role of these proteins during embryonic development, and most importantly for P. vitticeps, the molecular underpinnings of sex reversal.
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Epigénesis Genética/fisiología , Lagartos/fisiología , Procesos de Determinación del Sexo/fisiología , Temperatura , Animales , Ensamble y Desensamble de Cromatina/genética , Femenino , Gónadas/química , Histonas/análisis , Inmunohistoquímica/métodos , Inmunohistoquímica/veterinaria , Histona Demetilasas con Dominio de Jumonji/análisis , Lagartos/genética , Masculino , Metilación , Proteínas de Unión al ARN/análisis , Procesos de Determinación del Sexo/genéticaRESUMEN
Treatment options for COVID-19 remain limited, especially during the early or asymptomatic phase. Here, we report a novel SARS-CoV-2 viral replication mechanism mediated by interactions between ACE2 and the epigenetic eraser enzyme LSD1, and its interplay with the nuclear shuttling importin pathway. Recent studies have shown a critical role for the importin pathway in SARS-CoV-2 infection, and many RNA viruses hijack this axis to re-direct host cell transcription. LSD1 colocalized with ACE2 at the cell surface to maintain demethylated SARS-CoV-2 spike receptor-binding domain lysine 31 to promote virus-ACE2 interactions. Two newly developed peptide inhibitors competitively inhibited virus-ACE2 interactions, and demethylase access to significantly inhibit viral replication. Similar to some other predominantly plasma membrane proteins, ACE2 had a novel nuclear function: its cytoplasmic domain harbors a nuclear shuttling domain, which when demethylated by LSD1 promoted importin-α-dependent nuclear ACE2 entry following infection to regulate active transcription. A novel, cell permeable ACE2 peptide inhibitor prevented ACE2 nuclear entry, significantly inhibiting viral replication in SARS-CoV-2-infected cell lines, outperforming other LSD1 inhibitors. These data raise the prospect of post-exposure prophylaxis for SARS-CoV-2, either through repurposed LSD1 inhibitors or new, nuclear-specific ACE2 inhibitors.
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PURPOSE: G9a histone methyltransferase exerts oncogenic effects in several tumor types and its inhibition promotes anticancer effects. However, the impact on checkpoint inhibitor blockade response and the utility of G9a or its target genes as a biomarker is poorly studied. We aimed to examine whether G9a inhibition can augment the efficacy of checkpoint inhibitor blockade and whether LC3B, a G9a target gene, can predict treatment response. EXPERIMENTAL DESIGN: Clinical potential of LC3B as a biomarker of checkpoint inhibitor blockade was assessed using patient samples including tumor biopsies and circulating tumor cells from liquid biopsies. Efficacy of G9a inhibition to enhance checkpoint inhibitor blockade was examined using a mouse model. RESULTS: Patients with melanoma who responded to checkpoint inhibitor blockade were associated with not only a higher level of tumor LC3B but also a higher proportion of cells expressing LC3B. A higher expression of MAP1LC3B or LC3B protein was associated with longer survival and lower incidence of acquired resistance to checkpoint inhibitor blockade, suggesting LC3B as a potential predictive biomarker. We demonstrate that G9a histone methyltransferase inhibition is able to not only robustly induce LC3B level to augment the efficacy of checkpoint inhibitor blockade, but also induces melanoma cell death. CONCLUSIONS: Checkpoint inhibitor blockade response is limited to a subset of the patient population. These results have implications for the development of LC3B as a predictive biomarker of checkpoint inhibitor blockade to guide patient selection, as well as G9a inhibition as a strategy to extend the proportion of patients responding to immunotherapy.
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Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Inhibidores de Puntos de Control Inmunológico/farmacología , Melanoma/metabolismo , Animales , Biomarcadores de Tumor , Línea Celular Tumoral , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Melanoma/diagnóstico , Melanoma/tratamiento farmacológico , Melanoma/genética , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Células Neoplásicas Circulantes , Pronóstico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lysine specific demethylase 1 (LSD1) is a key epigenetic eraser enzyme implicated in cancer metastases and recurrence. Nuclear LSD1 phosphorylated at serine 111 (nLSD1p) has been shown to be critical for the development of breast cancer stem cells. Here we show that circulating tumor cells isolated from immunotherapy-resistant metastatic melanoma patients express higher levels of nLSD1p compared to responders, which is associated with co-expression of stem-like, mesenchymal genes. Targeting nLSD1p with selective nLSD1 inhibitors better inhibits the stem-like mesenchymal signature than traditional FAD-specific LSD1 catalytic inhibitors such as GSK2879552. We also demonstrate that nLSD1p is enriched in PD-1+CD8+ T cells from resistant melanoma patients and 4T1 immunotherapy-resistant mice. Targeting the LSD1p nuclear axis induces IFN-γ/TNF-α-expressing CD8+ T cell infiltration into the tumors of 4T1 immunotherapy-resistant mice, which is further augmented by combined immunotherapy. Underpinning these observations, nLSD1p is regulated by the key T cell exhaustion transcription factor EOMES in dysfunctional CD8+ T cells. EOMES co-exists with nLSD1p in PD-1+CD8+ T cells in resistant patients, and nLSD1p regulates EOMES nuclear dynamics via demethylation/acetylation switching of critical EOMES residues. Using novel antibodies to target these post-translational modifications, we show that EOMES demethylation/acetylation is reciprocally expressed in resistant and responder patients. Overall, we show for the first time that dual inhibition of metastatic cancer cells and re-invigoration of the immune system requires LSD1 inhibitors that target the nLSD1p axis.
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Reprogramación Celular/efectos de los fármacos , Reprogramación Celular/genética , Histona Demetilasas/genética , Neoplasias/etiología , Proteínas de Dominio T Box/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Animales , Biomarcadores , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Histona Demetilasas/metabolismo , Humanos , Inmunoterapia , Ratones , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/terapia , Proteínas de Dominio T Box/genética , Linfocitos T/inmunología , Resultado del TratamientoRESUMEN
DUSP6 is a dual-specificity phosphatase (DUSP) involved in breast cancer progression, recurrence, and metastasis. DUSP6 is predominantly cytoplasmic in HER2+ primary breast cancer cells, but the expression and subcellular localization of DUSPs, especially DUSP6, in HER2-positive circulating tumor cells (CTCs) is unknown. Here we used the DEPArray system to identify and isolate CTCs from metastatic triple negative breast cancer (TNBC) patients and performed single-cell NanoString analysis to quantify cancer pathway gene expression in HER2-positive and HER2-negative CTC populations. All TNBC patients contained HER2-positive CTCs. HER2-positive CTCs were associated with increased ERK1/ERK2 expression, which are direct DUSP6 targets. DUSP6 protein expression was predominantly nuclear in breast CTCs and the brain metastases but not pleura or lung metastases of TNBC patients. Therefore, nuclear DUSP6 may play a role in the association with cancer spreading in TNBC patients, including brain metastasis.
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Biomarcadores de Tumor , Neoplasias Encefálicas/secundario , Fosfatasa 6 de Especificidad Dual/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Antineoplásicos/farmacología , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Núcleo Celular/genética , Modelos Animales de Enfermedad , Fosfatasa 6 de Especificidad Dual/antagonistas & inhibidores , Fosfatasa 6 de Especificidad Dual/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Invasividad Neoplásica , Estadificación de Neoplasias , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Unión Proteica , Transporte de Proteínas , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Análisis de la Célula Individual , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/terapia , Ensayos Antitumor por Modelo de Xenoinjerto , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Macrophages play an important role in regulating the tumor microenvironment (TME). Here we show that classical (M1) macrophage polarization reduced expression of LSD1, nuclear REST corepressor 1 (CoREST), and the zinc finger protein SNAIL. The LSD1 inhibitor phenelzine targeted both the flavin adenine dinucleotide (FAD) and CoREST binding domains of LSD1, unlike the LSD1 inhibitor GSK2879552, which only targeted the FAD domain. Phenelzine treatment reduced nuclear demethylase activity and increased transcription and expression of M1-like signatures both in vitro and in a murine triple-negative breast cancer model. Overall, the LSD1 inhibitors phenelzine and GSK2879552 are useful tools for dissecting the contribution of LSD1 demethylase activity and the nuclear LSD1-CoREST complex to switching macrophage polarization programs. These findings suggest that inhibitors must have dual FAD and CoREST targeting abilities to successfully initiate or prime macrophages toward an anti-tumor M1-like phenotype in triple-negative breast cancer.
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Histona Demetilasas/metabolismo , Macrófagos/inmunología , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Diferenciación Celular , Proteínas Co-Represoras/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Flavina-Adenina Dinucleótido/metabolismo , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas/genética , Humanos , Activación de Macrófagos , Ratones , Proteínas del Tejido Nervioso/metabolismo , Fenelzina/farmacología , Células RAW 264.7 , ARN Interferente Pequeño/genética , Factores de Transcripción de la Familia Snail/metabolismo , Células TH1/inmunología , Neoplasias de la Mama Triple Negativas/inmunología , Microambiente TumoralRESUMEN
Immune reconstitution following hematopoietic stem cell transplantation (HSCT) is critical in preventing harmful sequelae in recipients with cytomegalovirus (CMV) infection. To understand the molecular mechanisms underlying immune reconstitution kinetics, we profiled the transcriptome-chromatin accessibility landscape of CMV-specific CD8+ T cells from HCST recipients with different immune reconstitution efficiencies. CMV-specific T cells from HSCT recipients with stable antiviral immunity expressed higher levels of interferon/defense response and cell cycle genes in an interconnected network involving PI3KCG, STAT5B, NFAT, RBPJ, and lower HDAC6, increasing chromatin accessibility at the enhancer regions of immune and T-cell receptor signaling pathway genes. By contrast, the transcriptional and epigenomic signatures of CMV-specific T cells from HSCT recipients with unstable immune reconstitution showed commonalities with T-cell responses in other nonresolving chronic infections. These signatures included higher levels of EGR and KLF factors that, along with lower JARID2 expression, maintained higher accessibility at promoter and CpG-rich regions of genes associated with apoptosis. Furthermore, epigenetic targeting via inhibition of HDAC6 or JARID2 enhanced the transcription of genes associated with differential responses, suggesting that drugs targeting epigenomic modifiers may have therapeutic potential for enhancing immune reconstitution in HSCT recipients. Taken together, these analyses demonstrate that transcription factors and chromatin modulators create different chromatin accessibility landscapes in T cells of HSCT recipients that not only affect immediate gene expression but also differentially prime cells for responses to additional signals. Epigenetic therapy may be a promising strategy to promote immune reconstitution in HSCT recipients.
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Reprogramación Celular/genética , Reprogramación Celular/inmunología , Epigénesis Genética , Reconstitución Inmune , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ensamble y Desensamble de Cromatina , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/etiología , Regulación de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Especificidad del Receptor de Antígeno de Linfocitos T , Factores de Transcripción/metabolismo , Transcriptoma , Receptores de Trasplantes , Replicación ViralRESUMEN
Memory T cells exhibit transcriptional memory and "remember" their previous pathogenic encounter to increase transcription on re-infection. However, how this transcriptional priming response is regulated is unknown. Here we performed global FAIRE-seq profiling of chromatin accessibility in a human T cell transcriptional memory model. Primary activation induced persistent accessibility changes, and secondary activation induced secondary-specific opening of previously less accessible regions associated with enhanced expression of memory-responsive genes. Increased accessibility occurred largely in distal regulatory regions and was associated with increased histone acetylation and relative H3.3 deposition. The enhanced re-stimulation response was linked to the strength of initial PKC-induced signalling, and PKC-sensitive increases in accessibility upon initial stimulation showed higher accessibility on re-stimulation. While accessibility maintenance was associated with ETS-1, accessibility at re-stimulation-specific regions was linked to NFAT, especially in combination with ETS-1, EGR, GATA, NFκB, and NR4A. Furthermore, NFATC1 was directly regulated by ETS-1 at an enhancer region. In contrast to the factors that increased accessibility, signalling from bHLH and ZEB family members enhanced decreased accessibility upon re-stimulation. Interplay between distal regulatory elements, accessibility, and the combined action of sequence-specific transcription factors allows transcriptional memory-responsive genes to "remember" their initial environmental encounter.
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Ensamble y Desensamble de Cromatina , Cromatina/genética , Memoria Inmunológica/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transcripción Genética , Acetilación , Sitios de Unión , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Factores de Transcripción GATA/metabolismo , Perfilación de la Expresión Génica , Histonas/metabolismo , Humanos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Factores de Transcripción NFATC/metabolismo , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
There is evidence that an aberrant tumour microenvironment (TME) facilitates cancer development, progression, and responses to treatment. While many of the mechanisms underlying the phenotype and cancer-promoting behaviour of the TME are unknown, epigenetic mechanisms in cancer cells and the TME are thought to play important roles. As a result, cancer profiling strategies for drug and biomarker development require a thorough understanding of both the epithelial tissue compartment and the TME. This review discusses recent advances in our understanding of how cancer epithelial cells interact with their microenvironment and how this knowledge can be exploited clinically.
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Neoplasias de la Mama/patología , Células Epiteliales/patología , Fibroblastos/patología , Células del Estroma/patología , Microambiente Tumoral , Animales , Neoplasias de la Mama/diagnóstico , Progresión de la Enfermedad , HumanosRESUMEN
Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4(+) T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells.
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Linfocitos T CD4-Positivos/metabolismo , Núcleo Celular/enzimología , Histonas/metabolismo , Memoria Inmunológica/genética , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Factor de Transcripción ReIA/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Cromatina/metabolismo , Regulación de la Expresión Génica , Histonas/química , Humanos , Células Jurkat , Fosforilación , Fosfoserina/metabolismo , Proteína Quinasa C-theta , Transducción de SeñalRESUMEN
Dual-specificity phosphatases (DUSPs) dephosphorylate threonine/serine and tyrosine residues on their substrates. Here we show that DUSP1, DUSP4, and DUSP6 are involved in epithelial-to-mesenchymal transition (EMT) and breast cancer stem cell (CSC) regulation. DUSP1, DUSP4, and DUSP6 are induced during EMT in a PKC pathway signal-mediated EMT model. We show for the first time that the key chromatin-associated kinase PKC-θ directly regulates a subset of DUSP family members. DUSP1, DUSP4, and DUSP6 globally but differentially co-exist with enhancer and permissive active histone post-translational modifications, suggesting that they play distinct roles in gene regulation in EMT/CSCs. We show that nuclear DUSP4 associates with the key acetyltransferase p300 in the context of the chromatin template and dynamically regulates the interplay between two key phosphorylation marks: the 1834 (active) and 89 (inhibitory) residues central to p300's acetyltransferase activity. Furthermore, knockdown with small-interfering RNAs (siRNAs) shows that DUSP4 is required for maintaining H3K27ac, a mark mediated by p300. DUSP1, DUSP4, and DUSP6 knockdown with siRNAs shows that they participate in the formation of CD44hi/CD24lo/EpCAM+ breast CSCs: DUSP1 knockdown reduces CSC formation, while DUSP4 and DUSP6 knockdown enhance CSC formation. Moreover, DUSP6 is overexpressed in patient-derived HER2+ breast carcinomas compared to benign mammary tissue. Taken together, these findings illustrate novel pleiotropic roles for DUSP family members in EMT and CSC regulation in breast cancer.
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Neoplasias de la Mama/patología , Fosfatasas de Especificidad Dual/metabolismo , Transición Epitelial-Mesenquimal , Células Madre Neoplásicas/patología , Biomarcadores de Tumor/deficiencia , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cromatina/metabolismo , Fosfatasas de Especificidad Dual/deficiencia , Fosfatasas de Especificidad Dual/genética , Proteína p300 Asociada a E1A/metabolismo , Epigenómica , Técnicas de Silenciamiento del Gen , Sitios Genéticos/genética , Histonas/química , Histonas/metabolismo , Humanos , Lisina/metabolismo , Células MCF-7 , Fosforilación , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de ProteínasRESUMEN
Alternative splicing of nuclear pre-mRNA is essential for generating protein diversity and regulating gene expression. While many immunologically relevant genes undergo alternative splicing, the role of regulated splicing in T cell immune responses is largely unexplored, and the signaling pathways and splicing factors that regulate alternative splicing in T cells are poorly defined. Here, we show using a combination of Jurkat T cells, human primary T cells, and ex vivo naïve and effector virus-specific T cells isolated after influenza A virus infection that SC35 phosphorylation is induced in response to stimulatory signals. We show that SC35 colocalizes with RNA polymerase II in activated T cells and spatially overlaps with H3K27ac and H3K4me3, which mark transcriptionally active genes. Interestingly, SC35 remains coupled to the active histone marks in the absence of continuing stimulatory signals. We show for the first time that nuclear PKC-θ co-exists with SC35 in the context of the chromatin template and is a key regulator of SC35 in T cells, directly phosphorylating SC35 peptide residues at RNA recognition motif and RS domains. Collectively, our findings suggest that nuclear PKC-θ is a novel regulator of the key splicing factor SC35 in T cells.
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BACKGROUND: Immunological memory is the ability of the immune system to respond more rapidly and effectively to previously encountered pathogens, a key feature of adaptive immunity. The capacity of memory T cells to "remember" previous cellular responses to specific antigens ultimately resides in their unique patterns of gene expression. Following re-exposure to an antigen, previously activated genes are transcribed more rapidly and robustly in memory T cells compared to their naïve counterparts. The ability for cells to remember past transcriptional responses is termed "adaptive transcriptional memory". RESULTS: Recent global epigenome studies suggest that epigenetic mechanisms are central to establishing and maintaining transcriptional memory, with elegant studies in model organisms providing tantalizing insights into the epigenetic programs that contribute to adaptive immunity. These epigenetic mechanisms are diverse, and include not only classical acetylation and methylation events, but also exciting and less well-known mechanisms involving histone structure, upstream signalling pathways, and nuclear localisation of genomic regions. CONCLUSIONS: Current global health challenges in areas such as tuberculosis and influenza demand not only more effective and safer vaccines, but also vaccines for a wider range of health priorities, including HIV, cancer, and emerging pathogens such as Ebola. Understanding the multi-layered epigenetic mechanisms that underpin the rapid recall responses of memory T cells following reactivation is a critical component of this development pathway.
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Epigénesis Genética , Memoria Inmunológica/genética , Linfocitos T/metabolismo , Transcripción Genética , Animales , Cromatina/metabolismo , Histonas/metabolismo , HumanosRESUMEN
Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor ß (TGF-ß) and the key inflammatory regulatory protein NF-κB. Chromatinized PKC-θ exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-θ-sensitive genes that are directly tethered to PKC-θ in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Transición Epitelial-Mesenquimal/genética , Isoenzimas/genética , Proteína Quinasa C/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Antígeno CD24/biosíntesis , Antígeno CD24/genética , Diferenciación Celular/genética , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Receptores de Hialuranos/biosíntesis , Receptores de Hialuranos/genética , Células MCF-7 , Subunidad p50 de NF-kappa B/biosíntesis , Subunidad p50 de NF-kappa B/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Células Madre Neoplásicas/patología , Proteína Quinasa C-theta , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Interferente Pequeño , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Transducción de Señal/genética , Esferoides Celulares/patología , Factor de Transcripción ReIA/biosíntesis , Factor de Transcripción ReIA/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Human Rhinovirus (HRV) infection results in shut down of essential cellular processes, in part through disruption of nucleocytoplasmic transport by cleavage of the nucleoporin proteins (Nups) that make up the host cell nuclear pore. Although the HRV genome encodes two proteases (2A and 3C) able to cleave host proteins such as Nup62, little is known regarding the specific contribution of each. Here we use transfected as well as HRV-infected cells to establish for the first time that 3C protease is most likely the mediator of cleavage of Nup153 during HRV infection, while Nup62 and Nup98 are likely to be targets of HRV2A protease. HRV16 3C protease was also able to elicit changes in the appearance and distribution of the nuclear speckle protein SC35 in transfected cells, implicating it as a key mediator of the mislocalisation of SC35 in HRV16-infected cells. In addition, 3C protease activity led to the redistribution of the nucleolin protein out of the nucleolus, but did not affect nuclear localisation of hnRNP proteins, implying that complete disruption of nucleocytoplasmic transport leading to relocalisation of hnRNP proteins from the nucleus to the cytoplasm in HRV-infected cells almost certainly requires 2A in addition to 3C protease. Thus, a specific role for HRV 3C protease in cleavage and mislocalisation of host cell nuclear proteins, in concert with 2A, is implicated for the first time in HRV pathogenesis.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virales/metabolismo , Proteasas Virales 3C , Transporte Activo de Núcleo Celular , Animales , Western Blotting , Células COS , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Chlorocebus aethiops , Cisteína Endopeptidasas/genética , Citoplasma/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Espacio Intracelular/metabolismo , Espacio Intracelular/virología , Microscopía Confocal , Proteínas de Complejo Poro Nuclear/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteolisis , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Rhinovirus/genética , Rhinovirus/metabolismo , Rhinovirus/fisiología , Ribonucleoproteínas/genética , Factores de Empalme Serina-Arginina , Transfección , Proteínas Virales/genética , NucleolinaRESUMEN
We recently provided the first description of a nuclear mechanism used by Protein Kinase C-theta (PKC-θ) to mediate T cell gene expression. In this mode, PKC-θ tethers to chromatin to form an active nuclear complex by interacting with proteins including RNA polymerase II, the histone kinase MSK-1, the demethylase LSD1, and the adaptor molecule 14-3-3ζ at regulatory regions of inducible immune response genes. Moreover, our genome-wide analysis identified many novel PKC-θ target genes and microRNAs implicated in T cell development, differentiation, apoptosis, and proliferation. We have expanded our ChIP-on-chip analysis and have now identified a transcription factor motif containing NF-κB binding sites that may facilitate recruitment of PKC-θ to chromatin at coding genes. Furthermore, NF-κB association with chromatin appears to be a prerequisite for the assembly of the PKC-θ active complex. In contrast, a distinct NF-κB-containing module appears to operate at PKC-θ targeted microRNA genes, and here NF-κB negatively regulates microRNA gene transcription. Our efforts are also focusing on distinguishing between the nuclear and cytoplasmic functions of PKCs to ascertain how these kinases may synergize their roles as both cytoplasmic signaling proteins and their functions on the chromatin template, together enabling rapid induction of eukaryotic genes. We have identified an alternative sequence within PKC-θ that appears to be important for nuclear translocation of this kinase. Understanding the molecular mechanisms used by signal transduction kinases to elicit specific and distinct transcriptional programs in T cells will enable scientists to refine current therapeutic strategies for autoimmune diseases and cancer.