RESUMEN
PirB is an inhibitory cell surface receptor particularly prominent on myeloid cells. PirB curtails the phenotypes of activated macrophages during inflammation or tumorigenesis, but its functions in macrophage homeostasis are obscure. To elucidate PirB-related functions in macrophages at steady-state, we generated and compared single-cell RNA-sequencing (scRNAseq) datasets obtained from myeloid cell subsets of wild type (WT) and PirB-deficient knockout (PirB KO) mice. To facilitate this analysis, we developed a novel approach to clustering parameter optimization called "Cluster Similarity Scoring and Distinction Index" (CaSSiDI). We demonstrate that CaSSiDI is an adaptable computational framework that facilitates tandem analysis of two scRNAseq datasets by optimizing clustering parameters. We further show that CaSSiDI offers more advantages than a standard Seurat analysis because it allows direct comparison of two or more independently clustered datasets, thereby alleviating the need for batch-correction while identifying the most similar and different clusters. Using CaSSiDI, we found that PirB is a novel regulator of Cebpb expression that controls the generation of Ly6Clo patrolling monocytes and the expansion properties of peritoneal macrophages. PirB's effect on Cebpb is tissue-specific since it was not observed in splenic red pulp macrophages (RPMs). However, CaSSiDI revealed a segregation of the WT RPM population into a CD68loIrf8+ "neuronal-primed" subset and an CD68hiFtl1+ "iron-loaded" subset. Our results establish the utility of CaSSiDI for single-cell assay analyses and the determination of optimal clustering parameters. Our application of CaSSiDI in this study has revealed previously unknown roles for PirB in myeloid cell populations. In particular, we have discovered homeostatic functions for PirB that are related to Cebpb expression in distinct macrophage subsets.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT , Macrófagos , Receptores Inmunológicos , Análisis de la Célula Individual , Animales , Ratones , Macrófagos/metabolismo , Monocitos/metabolismo , Células Mieloides/metabolismo , Receptores de Superficie Celular , Receptores Inmunológicos/metabolismo , Análisis de la Célula Individual/métodos , Proteína beta Potenciadora de Unión a CCAAT/metabolismoRESUMEN
Cholinergic nerves are involved in tumor progression and dissemination. In contrast to other visceral tissues, cholinergic innervation in the hepatic parenchyma is poorly detected. It remains unclear whether there is any form of cholinergic regulation of liver cancer. Here, we show that cholinergic T cells curtail the development of liver cancer by supporting antitumor immune responses. In a mouse multihit model of hepatocellular carcinoma (HCC), we observed activation of the adaptive immune response and induction of two populations of CD4+ T cells expressing choline acetyltransferase (ChAT), including regulatory T cells and dysfunctional PD-1+ T cells. Tumor antigens drove the clonal expansion of these cholinergic T cells in HCC. Genetic ablation of Chat in T cells led to an increased prevalence of preneoplastic cells and exacerbated liver cancer due to compromised antitumor immunity. Mechanistically, the cholinergic activity intrinsic in T cells constrained Ca2+-NFAT signaling induced by T cell antigen receptor engagement. Without this cholinergic modulation, hyperactivated CD25+ T regulatory cells and dysregulated PD-1+ T cells impaired HCC immunosurveillance. Our results unveil a previously unappreciated role for cholinergic T cells in liver cancer immunobiology.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Ratones , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Receptor de Muerte Celular Programada 1/genética , Monitorización Inmunológica , Linfocitos T Reguladores/patologíaRESUMEN
Current vaccine efforts to combat SARS-CoV-2 are focused on the whole spike protein administered as mRNA, viral vector, or protein subunit. However, the SARS-CoV-2 receptor-binding domain (RBD) is the immunodominant portion of the spike protein, accounting for 90% of serum neutralizing activity. In this study, we constructed several versions of RBD and together with aluminum hydroxide or DDA (dimethyldioctadecylammonium bromide)/TDB (d-(+)-trehalose 6,6'-dibehenate) adjuvant evaluated immunogenicity in mice. We generated human angiotensin-converting enzyme 2 knock-in mice to evaluate vaccine efficacy in vivo following viral challenge. We found that 1) subdomain (SD)1 was essential for the RBD to elicit maximal immunogenicity; 2) RBDSD1 produced in mammalian HEK cells elicited better immunogenicity than did protein produced in insect or yeast cells; 3) RBDSD1 combined with the CD4 Th1 adjuvant DDA/TDB produced higher neutralizing Ab responses and stronger CD4 T cell responses than did aluminum hydroxide; 4) addition of monomeric human Fc receptor to RBDSD1 (RBDSD1Fc) significantly enhanced immunogenicity and neutralizing Ab titers; 5) the Beta version of RBDSD1Fc provided a broad range of cross-neutralization to multiple antigenic variants of concern, including Omicron; and 6) the Beta version of RBDSD1Fc with DDA/TDB provided complete protection against virus challenge in the knock-in mouse model. Thus, we have identified an optimized RBD-based subunit vaccine suitable for clinical trials.
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COVID-19 , Vacunas Virales , Humanos , Animales , Ratones , SARS-CoV-2 , Vacunas contra la COVID-19 , Hidróxido de Aluminio , Glicoproteína de la Espiga del Coronavirus , Vacunas de Subunidad , Anticuerpos Antivirales , Anticuerpos Neutralizantes , MamíferosRESUMEN
Angioimmunoblastic T cell lymphoma (AITL) is a peripheral T cell lymphoma that originates from T follicular helper (Tfh) cells and exhibits a prominent tumor microenvironment (TME). IDH2 and TET2 mutations co-occur frequently in AITL, but their contribution to tumorigenesis is poorly understood. We developed an AITL mouse model that is driven by Idh2 and Tet2 mutations. Malignant Tfh cells display aberrant transcriptomic and epigenetic programs that impair TCR signaling. Neoplastic Tfh cells bearing combined Idh2 and Tet2 mutations show altered cross-talk with germinal center B cells that promotes B cell clonal expansion while decreasing Fas-FasL interaction and reducing B cell apoptosis. The plasma cell count and angiogenesis are also increased in the Idh2-mutated tumors, implying a major relationship between Idh2 mutation and the characteristic AITL TME. Our mouse model recapitulates several features of human IDH2-mutated AITL and provides a rationale for exploring therapeutic targeting of Tfh-TME cross-talk for AITL patients.
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Dioxigenasas , Linfadenopatía Inmunoblástica , Linfoma de Células T , Animales , Humanos , Ratones , Dioxigenasas/genética , Proteínas de Unión al ADN/genética , Linfadenopatía Inmunoblástica/genética , Isocitrato Deshidrogenasa/genética , Linfoma de Células T/genética , Mutación , Células T Auxiliares Foliculares/patología , Linfocitos T Colaboradores-Inductores , Microambiente Tumoral/genéticaRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS) in which Th17 cells have a crucial but unclear function. Here we show that choline acetyltransferase (ChAT), which synthesizes acetylcholine (ACh), is a critical driver of pathogenicity in EAE. Mice with ChAT-deficient Th17 cells resist disease progression and show reduced brain-infiltrating immune cells. ChAT expression in Th17 cells is linked to strong TCR signaling, expression of the transcription factor Bhlhe40, and increased Il2, Il17, Il22, and Il23r mRNA levels. ChAT expression in Th17 cells is independent of IL21r signaling but dampened by TGFß, implicating ChAT in controlling the dichotomous nature of Th17 cells. Our study establishes a cholinergic program in which ACh signaling primes chronic activation of Th17 cells, and thereby constitutes a pathogenic determinant of EAE. Our work may point to novel targets for therapeutic immunomodulation in MS.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Ratones , Animales , Células Th17 , Virulencia , Colinérgicos , Esclerosis Múltiple/genética , Acetilcolina/metabolismo , Ratones Endogámicos C57BL , Diferenciación CelularRESUMEN
Type I and II interferons (IFNs) stimulate pro-inflammatory programs that are critical for immune activation, but also induce immune-suppressive feedback circuits that impede control of cancer growth. Here, we sought to determine how these opposing programs are differentially induced. We demonstrated that the transcription factor interferon regulatory factor 2 (IRF2) was expressed by many immune cells in the tumor in response to sustained IFN signaling. CD8+ T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor and instead enabled sustained effector functions that promoted long-term tumor control and increased responsiveness to immune checkpoint and adoptive cell therapies. The long-term tumor control by IRF2-deficient CD8+ T cells required continuous integration of both IFN-I and IFN-II signals. Thus, IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a potential target to enhance cancer control.
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Interferón Tipo I , Neoplasias , Humanos , Factor 2 Regulador del Interferón/genética , Linfocitos T CD8-positivos , Factores de Transcripción , Agotamiento de Células T , Neoplasias/patologíaRESUMEN
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here we identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), which has antitumor growth activity in TNBC. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses before and after MS023 treatment is a functional biomarker and determinant of response, and these observations extend to a panel of human-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA, which is derived, at least in part, from inverted repeat Alu elements. Together, our results represent a shift in understanding the antitumor mechanism of type I PRMT inhibitors and provide a rationale and biomarker approach for the clinical development of type I PRMT inhibitors.
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Proteína-Arginina N-Metiltransferasas , Neoplasias de la Mama Triple Negativas , Biomarcadores , Línea Celular Tumoral , Humanos , Interferones/uso terapéutico , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Patient-derived tumor organoids (PDOs) are a highly promising preclinical model that recapitulates the histology, gene expression, and drug response of the donor patient tumor. Currently, PDO culture relies on basement-membrane extract (BME), which suffers from batch-to-batch variability, the presence of xenogeneic compounds and residual growth factors, and poor control of mechanical properties. Additionally, for the development of new organoid lines from patient-derived xenografts, contamination of murine host cells poses a problem. We propose a nanofibrillar hydrogel (EKGel) for the initiation and growth of breast cancer PDOs. PDOs grown in EKGel have histopathologic features, gene expression, and drug response that are similar to those of their parental tumors and PDOs in BME. In addition, EKGel offers reduced batch-to-batch variability, a range of mechanical properties, and suppressed contamination from murine cells. These results show that EKGel is an improved alternative to BME matrices for the initiation, growth, and maintenance of breast cancer PDOs.
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Neoplasias de la Mama , Organoides , Animales , Biomimética , Neoplasias de la Mama/patología , Femenino , Humanos , Hidrogeles/metabolismo , Hidrogeles/farmacología , Ratones , Organoides/metabolismoRESUMEN
T lymphocytes are increasingly implicated in pain signaling. A subset of T lymphocytes, termed TChAT, express the rate-limiting enzyme for acetylcholine (ACh) production, choline acetyltransferase (ChAT), and mediate numerous physiological functions. Given that cholinergic signaling has long been known to modulate pain processing and is the basis for several analgesics used clinically, we asked whether TChAT could be the intersection between T lymphocyte and cholinergic mediation of pain signaling. In this study, we used a mouse gene knockout strategy to ablate ChAT specifically from T lymphocytes and examined the development and expression of mechanical and thermal hypersensitivity in a spared nerve injury (SNI) mouse model of neuropathic pain. We found that mice with ChAT knockout in T cells (floxed Chat plus CD4-Cre recombinase) did not differ from control mice with intact ChAT (floxed Chat, but no Cre recombinase) in their expression of mechanical sensitivity before or after injury. Similarly, thermal sensitivity was unaffected after injury, with control mice expressing similar patterns of thermal preference to mice whose T cells do not express ChAT. Our experiments demonstrate that cholinergic signaling initiated by T lymphocytes neither dampens nor exacerbates the expression of mechanical or thermal sensitivity in neuropathic mice. Thus, while both cholinergic signaling and T lymphocytes have established roles in modulating pain phenotypes, it is not cholinergic signaling initiated by T lymphocytes that drive this. Our findings will help to narrow in on which aspects of T-cell modulation may prove useful as therapies.
Asunto(s)
Neuralgia , Linfocitos T , Acetilcolina/metabolismo , Animales , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Colinérgicos/metabolismo , Ratones , Neuralgia/metabolismo , Linfocitos T/metabolismoRESUMEN
The tumor suppressor PTEN is disrupted in a large proportion of cancers, including in HER2-positive breast cancer, where its loss is associated with resistance to therapy. Upon genotoxic stress, ataxia telangiectasia mutated (ATM) is activated and phosphorylates PTEN on residue 398. To elucidate the physiological role of this molecular event, we generated and analyzed knock-in mice expressing a mutant form of PTEN that cannot be phosphorylated by ATM (PTEN-398A). This mutation accelerated tumorigenesis in a model of HER2-positive breast cancer. Mammary tumors in bi-transgenic mice carrying MMTV-neu and Pten398A were characterized by DNA damage accumulation but reduced apoptosis. Mechanistically, phosphorylation of PTEN at position 398 is essential for the proper activation of the S phase checkpoint controlled by the PI3K-p27Kip1-CDK2 axis. Moreover, we linked these defects to the impaired ability of the PTEN-398A protein to relocalize to the plasma membrane in response to genotoxic stress. Altogether, our results uncover a novel role for ATM-dependent PTEN phosphorylation in the control of genomic stability, cell cycle progression, and tumorigenesis.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias de la Mama/genética , Puntos de Control del Ciclo Celular/genética , Fosfohidrolasa PTEN/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Carcinogénesis , Ciclo Celular , Femenino , Humanos , Neoplasias Mamarias Animales , RatonesRESUMEN
In order to sustain proficient life-long hematopoiesis, hematopoietic stem cells (HSCs) must possess robust mechanisms to preserve their quiescence and genome integrity. DNA-damaging stress can perturb HSC homeostasis by affecting their survival, self-renewal, and differentiation. Ablation of the kinase ataxia telangiectasia mutated (ATM), a master regulator of the DNA damage response, impairs HSC fitness. Paradoxically, we show here that loss of a single allele of Atm enhances HSC functionality in mice. To explain this observation, we explored a possible link between ATM and the tumor suppressor phosphatase and tensin homolog (PTEN), which also regulates HSC function. We generated and analyzed a knockin mouse line (PtenS398A/S398A), in which PTEN cannot be phosphorylated by ATM. Similar to Atm+/-, PtenS398A/S398A HSCs have enhanced hematopoietic reconstitution ability, accompanied by resistance to apoptosis induced by genotoxic stress. Single-cell transcriptomic analyses and functional assays revealed that dormant PtenS398A/S398A HSCs aberrantly tolerate elevated mitochondrial activity and the accumulation of reactive oxygen species, which are normally associated with HSC priming for self-renewal or differentiation. Our results unveil a molecular connection between ATM and PTEN, which couples the response to genotoxic stress and dormancy in HSCs.
Asunto(s)
Apoptosis , Diferenciación Celular , Daño del ADN , Células Madre Hematopoyéticas/metabolismo , Fosfohidrolasa PTEN/metabolismo , Sustitución de Aminoácidos , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ratones , Ratones Transgénicos , Mutación Missense , Fosfohidrolasa PTEN/genéticaRESUMEN
Triple negative breast cancer (TNBC) is a deadly form of breast cancer due to the development of resistance to chemotherapy affecting over 30% of patients. New therapeutics and companion biomarkers are urgently needed. Recognizing the elevated expression of glucose transporter 1 (GLUT1, encoded by SLC2A1) and associated metabolic dependencies in TNBC, we investigated the vulnerability of TNBC cell lines and patient-derived samples to GLUT1 inhibition. We report that genetic or pharmacological inhibition of GLUT1 with BAY-876 impairs the growth of a subset of TNBC cells displaying high glycolytic and lower oxidative phosphorylation (OXPHOS) rates. Pathway enrichment analysis of gene expression data suggests that the functionality of the E2F pathway may reflect to some extent OXPHOS activity. Furthermore, the protein levels of retinoblastoma tumor suppressor (RB1) strongly correlate with the degree of sensitivity to GLUT1 inhibition in TNBC, where RB1-negative cells are insensitive to GLUT1 inhibition. Collectively, our results highlight a strong and targetable RB1-GLUT1 metabolic axis in TNBC and warrant clinical evaluation of GLUT1 inhibition in TNBC patients stratified according to RB1 protein expression levels.
Asunto(s)
Transportador de Glucosa de Tipo 1/antagonistas & inhibidores , Transportador de Glucosa de Tipo 1/metabolismo , Proteínas de Unión a Retinoblastoma/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor , Neoplasias de la Mama/metabolismo , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 1/genética , Humanos , Ratones , Fosforilación Oxidativa , Proteómica , Pirazoles/farmacología , Piridinas/farmacología , Quinolinas , ARN Mensajero/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors for which there is currently no effective treatment. Some of these tumors combine gain-of-function mutations in ACVR1, PIK3CA, and histone H3-encoding genes. The oncogenic mechanisms of action of ACVR1 mutations are currently unknown. Using mouse models, we demonstrate that Acvr1G328V arrests the differentiation of oligodendroglial lineage cells, and cooperates with Hist1h3bK27M and Pik3caH1047R to generate high-grade diffuse gliomas. Mechanistically, Acvr1G328V upregulates transcription factors which control differentiation and DIPG cell fitness. Furthermore, we characterize E6201 as a dual inhibitor of ACVR1 and MEK1/2, and demonstrate its efficacy toward tumor cells in vivo. Collectively, our results describe an oncogenic mechanism of action for ACVR1 mutations, and suggest therapeutic strategies for DIPGs.
Asunto(s)
Receptores de Activinas Tipo I/química , Receptores de Activinas Tipo I/genética , Neoplasias Encefálicas/patología , Glioma/patología , Mutación , Receptores de Activinas Tipo I/antagonistas & inhibidores , Receptores de Activinas Tipo I/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Femenino , Glioma/tratamiento farmacológico , Glioma/genética , Histonas/genética , Histonas/metabolismo , Humanos , Lactonas/farmacología , Masculino , Ratones Transgénicos , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Neuroglía/metabolismo , Neuroglía/patología , Oligodendroglía/patología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/metabolismoRESUMEN
Regulatory T cells (Tregs) maintain immune homeostasis and prevent autoimmunity. Serine stimulates glutathione (GSH) synthesis and feeds into the one-carbon metabolic network (1CMet) essential for effector T cell (Teff) responses. However, serine's functions, linkage to GSH, and role in stress responses in Tregs are unknown. Here, we show, using mice with Treg-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that GSH loss in Tregs alters serine import and synthesis and that the integrity of this feedback loop is critical for Treg suppressive capacity. Although Gclc ablation does not impair Treg differentiation, mutant mice exhibit severe autoimmunity and enhanced anti-tumor responses. Gclc-deficient Tregs show increased serine metabolism, mTOR activation, and proliferation but downregulated FoxP3. Limitation of cellular serine in vitro and in vivo restores FoxP3 expression and suppressive capacity of Gclc-deficient Tregs. Our work reveals an unexpected role for GSH in restricting serine availability to preserve Treg functionality.
Asunto(s)
Glutatión/metabolismo , Serina/metabolismo , Linfocitos T Reguladores/metabolismo , Animales , RatonesRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Myeloid cells contribute to tumor progression, but how the constellation of receptors they express regulates their functions within the tumor microenvironment (TME) is unclear. We demonstrate that Fcmr (Toso), the putative receptor for soluble IgM, modulates myeloid cell responses to cancer. In a syngeneic melanoma model, Fcmr ablation in myeloid cells suppressed tumor growth and extended mouse survival. Fcmr deficiency increased myeloid cell population density in this malignancy and enhanced anti-tumor immunity. Single-cell RNA sequencing of Fcmr-deficient tumor-associated mononuclear phagocytes revealed a unique subset with enhanced antigen processing/presenting properties. Conversely, Fcmr activity negatively regulated the activation and migratory capacity of myeloid cells in vivo, and T cell activation by bone marrow-derived dendritic cells in vitro. Therapeutic targeting of Fcmr during oncogenesis decreased tumor growth when used as a single agent or in combination with anti-PD-1. Thus, Fcmr regulates myeloid cell activation within the TME and may be a potential therapeutic target.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas Portadoras/metabolismo , Melanoma Experimental/inmunología , Proteínas de la Membrana/metabolismo , Monocitos/inmunología , Neoplasias Cutáneas/inmunología , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinogénesis/efectos de los fármacos , Carcinogénesis/inmunología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Línea Celular Tumoral/trasplante , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Femenino , Activación de Linfocitos/inmunología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/mortalidad , Melanoma Experimental/patología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Análisis de Supervivencia , Linfocitos T/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Cancer cells have higher reactive oxygen species (ROS) than normal cells, due to genetic and metabolic alterations. An emerging scenario is that cancer cells increase ROS to activate protumorigenic signaling while activating antioxidant pathways to maintain redox homeostasis. Here we show that, in basal-like and BRCA1-related breast cancer (BC), ROS levels correlate with the expression and activity of the transcription factor aryl hydrocarbon receptor (AhR). Mechanistically, ROS triggers AhR nuclear accumulation and activation to promote the transcription of both antioxidant enzymes and the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). In a mouse model of BRCA1-related BC, cancer-associated AhR and AREG control tumor growth and production of chemokines to attract monocytes and activate proangiogenic function of macrophages in the tumor microenvironment. Interestingly, the expression of these chemokines as well as infiltration of monocyte-lineage cells (monocyte and macrophages) positively correlated with ROS levels in basal-like BC. These data support the existence of a coordinated link between cancer-intrinsic ROS regulation and the features of tumor microenvironment. Therapeutically, chemical inhibition of AhR activity sensitizes human BC models to Erlotinib, a selective EGFR tyrosine kinase inhibitor, suggesting a promising combinatorial anticancer effect of AhR and EGFR pathway inhibition. Thus, AhR represents an attractive target to inhibit redox homeostasis and modulate the tumor promoting microenvironment of basal-like and BRCA1-associated BC.
Asunto(s)
Anfirregulina/genética , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Receptores de Hidrocarburo de Aril/genética , Adulto , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Receptores ErbB/genética , Clorhidrato de Erlotinib/administración & dosificación , Femenino , Regulación Neoplásica de la Expresión Génica , Homeostasis/genética , Humanos , Ratones , Persona de Mediana Edad , Oxidación-Reducción/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Microambiente Tumoral/genéticaRESUMEN
Although widely studied as a neurotransmitter, T cell-derived acetylcholine (ACh) has recently been reported to play an important role in regulating immunity. However, the role of lymphocyte-derived ACh in viral infection is unknown. Here, we show that the enzyme choline acetyltransferase (ChAT), which catalyzes the rate-limiting step of ACh production, is robustly induced in both CD4+ and CD8+ T cells during lymphocytic choriomeningitis virus (LCMV) infection in an IL-21-dependent manner. Deletion of Chat within the T cell compartment in mice ablated vasodilation in response to infection, impaired the migration of antiviral T cells into infected tissues, and ultimately compromised the control of chronic LCMV clone 13 infection. Our results reveal a genetic proof of function for ChAT in T cells during viral infection and identify a pathway of T cell migration that sustains antiviral immunity.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Colina O-Acetiltransferasa/inmunología , Interleucinas/inmunología , Coriomeningitis Linfocítica/inmunología , Animales , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/enzimología , Movimiento Celular , Colina O-Acetiltransferasa/genética , Femenino , Activación de Linfocitos , Virus de la Coriomeningitis Linfocítica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , VasodilataciónRESUMEN
Wnt signaling, named after the secreted proteins that bind to cell surface receptors to activate the pathway, plays critical roles both in embryonic development and the maintenance of homeostasis in many adult tissues. Two particularly important cellular programs orchestrated by Wnt signaling are proliferation and stem cell self-renewal. Constitutive activation of the Wnt pathway resulting from mutation or improper modulation of pathway components contributes to cancer development in various tissues. Colon cancers frequently bear inactivating mutations of the adenomatous polyposis coli (APC) gene, whose product is an important component of the destruction complex that regulates ß-catenin levels. Stabilization and nuclear localization of ß-catenin result in the expression of a panel of Wnt target genes. We previously showed that Mule/Huwe1/Arf-BP1 (Mule) controls murine intestinal stem and progenitor cell proliferation by modulating the Wnt pathway via c-Myc. Here we extend our investigation of Mule's influence on oncogenesis by showing that Mule interacts directly with ß-catenin and targets it for degradation under conditions of hyperactive Wnt signaling. Our findings suggest that Mule uses various mechanisms to fine-tune the Wnt pathway and provides multiple safeguards against tumorigenesis.
Asunto(s)
Proteínas Supresoras de Tumor/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Vía de Señalización Wnt , beta Catenina/antagonistas & inhibidores , Proteína de la Poliposis Adenomatosa del Colon/deficiencia , Animales , Proteína Axina/biosíntesis , Proteína Axina/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Neoplasias del Colon/metabolismo , Ciclina D1/biosíntesis , Ciclina D1/genética , Regulación hacia Abajo , Genes APC , Genes Supresores de Tumor , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Proteínas de Neoplasias/fisiología , Organoides/metabolismo , Organoides/ultraestructura , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteolisis , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Recombinantes/metabolismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
T-cell proliferation is regulated by ubiquitination but the underlying molecular mechanism remains obscure. Here we report that Lys-48-linked ubiquitination of the transcription factor KLF4 mediated by the E3 ligase Mule promotes T-cell entry into S phase. Mule is elevated in T cells upon TCR engagement, and Mule deficiency in T cells blocks proliferation because KLF4 accumulates and drives upregulation of its transcriptional targets E2F2 and the cyclin-dependent kinase inhibitors p21 and p27. T-cell-specific Mule knockout (TMKO) mice develop exacerbated experimental autoimmune encephalomyelitis (EAE), show impaired generation of antigen-specific CD8+ T cells with reduced cytokine production, and fail to clear LCMV infections. Thus, Mule-mediated ubiquitination of the novel substrate KLF4 regulates T-cell proliferation, autoimmunity and antiviral immune responses in vivo.