RESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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
The massage industry has been in the market for more than thousands of years. Consumers purchase massage services to treat illnesses, alleviate pain, or improve well-being. Despite the popularity of this industry and the benefits it entails, consumers' preferences vary and massage parlors' stakeholders have inconsistent market segmentation. Therefore, the purpose of this study was to investigate consumer preferences toward massage services offered by massage parlors in Indonesia through conjoint analysis. Conjoint analysis' orthogonal design concentrated on stimuli preferences and it was further supported by generating 1.000 consistency and reliability based on Kendall Tau's holdout. A total of 212 respondents answered the online questionnaire voluntarily. The results showed that the Google rating was the most important attribute (68.5%), followed by the gender of the massage therapist (12.4%), the type of massage (9.6%), the type of room (4.5%), the duration (3.6%) and the price (1.4%). Moreover, this research assessed 27 stimuli and found that the best combination was employing female massage therapists, IDR 100k-150k price every session, 90 minutes massage duration, couple room with two beds, acupuncture and cupping massage type, and massage parlors with greater than 4.6 Google review rating. This present research was one of the first studies that explored unique and holistic massage services through conjoint analysis. Unlike the previous studies that only focused on one massage service attribute or did not provide specific levels for evaluated attributes, the conjoint's orthogonal design delivered a new perception of prioritizing both consumers and the business side as some would only focus on one or the other. Also, the findings could be useful for massage parlors' stakeholders in developing marketing strategies, enhancing operational strategies, and promoting customer satisfaction. Marketing tactics such as promotional discounts would inspire customers to provide an optimistic Google review. Business owners were encouraged to focus on giving the best experience starting with a well-trained massage therapist, clean and hygienic rooms, and quality materials. These theoretical and practical implications aid in building the massage parlor's credibility that could be perceived positively by consumers.
Asunto(s)
Comportamiento del Consumidor , Masaje , Masaje/métodos , Humanos , Femenino , Masculino , Adulto , Indonesia , Encuestas y Cuestionarios , Persona de Mediana Edad , Adulto JovenRESUMEN
Recently discovered tet(X) gene variants have provided new insights into microbial antibiotic resistance mechanisms and their potential consequences for public health. This study focused on detection, analysis, and characterization of Tet(X4)-positive Enterobacterales from the gut microbiota of a healthy cohort of individuals in Singapore using cultivation-dependent and cultivation-independent approaches. Twelve Tet(X4)-positive Enterobacterales strains that were previously obtained from the cohort were fully genome-sequenced and comparatively analyzed. A metagenomic sequencing (MS) data set of the same samples was mined for contigs that harbored the tet(X4) resistance gene. The sequences of tet(X4)-containing contigs and plasmids sequences were compared. The presence of the resistance genes floR and estT (previously annotated as catD) was detected in the same cassette in 10 and 12 out of the 12 tet(X4)-carrying plasmids, respectively. MS detected tet(X4)-containing contigs in 2 out of the 109 subjects, while cultivation-dependent analysis previously reported a prevalence of 10.1%. The tet(X4)-containing sequences assembled from MS data are relatively short (~14 to 33 kb) but show high similarity to the respective plasmid sequences of the isolates. Our findings show that MS can complement efforts in the surveillance of antibiotic resistance genes for clinical samples, while it has a lower sensitivity than a cultivation-based method when the target organism has a low abundance. Further optimization is required if MS is to be utilized in antibiotic resistance surveillance.IMPORTANCEThe global rise in antibiotic resistance makes it necessary to develop and apply new approaches to detect and monitor the prevalence of antibiotic resistance genes in human populations. In this regard, of particular interest are resistances against last-resort antibiotics, such as tigecycline. In this study, we show that metagenomic sequencing can help to detect high abundance of the tigecycline resistance gene tet(X4) in fecal samples from a cohort of healthy human subjects. However, cultivation-based approaches currently remain the most reliable and cost-effective method for detection of antibiotic-resistant bacteria.
Asunto(s)
Gammaproteobacteria , Metagenoma , Humanos , Tigeciclina , Voluntarios Sanos , Antibacterianos/farmacología , Plásmidos/genética , Pruebas de Sensibilidad MicrobianaRESUMEN
A remarkable feature of the adaptive immune system is the speed at which small numbers of antigen-specific lymphocytes can mediate a successful immune response. Rapid expansion of T and B lymphocyte clones that have receptors specific for a particular antigen is one of the primary means by which a swift response is generated. Although much of this clonal expansion is caused by the division of antigen-specific cells, here we demonstrate an additional mechanism by which the pool of effector T cells against a viral infection can quickly enlarge. Our data show that virus-specific CD8+ cytotoxic T lymphocytes (CTL) can transfer their T cell receptors (TCR) to recipient CTL of an unrelated specificity that, as a consequence, gain the antigen specificity of the donor T cell. This process occurs within minutes via membrane exchange and results in the recipient CTL acquiring the ability to recognize and eliminate cells targeted by the donor TCR, while still retaining the antigen specificity of its own TCR. Such receptor sharing allows rapid, proliferation-independent expansion of virus-specific T cell clones of low frequency and plays a highly significant antiviral role that can protect the host from an otherwise lethal infection.
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Virus de la Ectromelia/inmunología , Ectromelia Infecciosa/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Membrana Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Ectromelia Infecciosa/sangre , Epítopos/inmunología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Carga ViralRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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
Endothelins regulate cellular functions in the mammalian brain through the endothelin receptors A and B (EDNRA and EDNRB). In this study, we investigated the role of EDNRB on cell proliferation in the cerebellum by using the spotting lethal (sl) rat, which carries a naturally occurring deletion in the EDNRB gene. Proliferating cells in the three genotypes, wild-type (+/+), heterozygous (+/sl) and homozygous mutant (sl/sl) rats were labelled by intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) at postnatal day 2. The density of BrdU-positive cells (per mm(2)) in the external germinal layer of sl/sl rats (Mean +/- SEM, 977 +/- 388) was significantly reduced compared to +/+ (4915 +/- 631) and +/sl (2304 +/- 557) rats. Subsequently, we examined the effects of EDNRB mutation on neural apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling assay. This showed that the density of apoptotic cells in the cerebella of sl/sl rats (9.3 +/- 0.5/mm(2)) was significantly more increased than +/+ rats (4 +/- 0.7). The expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were measured with standard ELISA, but were unchanged in all genotypes. These results suggest that ENDRB mediates neural proliferation and have anti-apoptotic effects in the cerebellum of the postnatal rat, and that these effects are independent of changes in the expression of BDNF and GDNF. Our findings will lead to better understanding of the morphological changes in the cerebellum of Hirschsprung's disease patients with congenital EDNRB mutation.
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Apoptosis , Cerebelo/citología , Cerebelo/metabolismo , Neuronas/citología , Receptor de Endotelina B/deficiencia , Células Madre/citología , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Bromodesoxiuridina/metabolismo , Recuento de Células , Núcleo Celular/metabolismo , Proliferación Celular , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Etiquetado Corte-Fin in Situ , Mutación/genética , Neuronas/metabolismo , Ratas , Ratas Wistar , Receptor de Endotelina B/metabolismo , Células Madre/metabolismoRESUMEN
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.
Asunto(s)
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
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.