RESUMEN
A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Resistencia a Antineoplásicos/inmunología , Neoplasias/tratamiento farmacológico , Proclorperazina/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Presentación de Antígeno/efectos de los fármacos , Biopsia , Cetuximab/farmacología , Sistemas de Liberación de Medicamentos/métodos , Resistencia a Antineoplásicos/genética , Endocitosis/efectos de los fármacos , Endocitosis/inmunología , Xenoinjertos , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células MCF-7 , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Neoplasias/genética , Neoplasias/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Trastuzumab/farmacologíaRESUMEN
Proteotypes, like genotypes, have been found to vary between individuals in several studies, but consistent molecular functional traits across studies remain to be quantified. In a meta-analysis of 11 proteomics datasets from humans and mice, we use co-variation of proteins in known functional modules across datasets and individuals to obtain a consensus landscape of proteotype variation. We find that individuals differ considerably in both protein complex abundances and stoichiometry. We disentangle genetic and environmental factors impacting these metrics, with genetic sex and specific diets together explaining 13.5% and 11.6% of the observed variation of complex abundance and stoichiometry, respectively. Sex-specific differences, for example, include various proteins and complexes, where the respective genes are not located on sex-specific chromosomes. Diet-specific differences, added to the individual genetic backgrounds, might become a starting point for personalized proteotype modulation toward desired features.
Asunto(s)
Bases de Datos de Proteínas , Interacción Gen-Ambiente , Genotipo , Caracteres Sexuales , Células A549 , Animales , Femenino , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Células K562 , Células MCF-7 , Masculino , Ratones , ProteómicaRESUMEN
While knowledge of protein-protein interactions (PPIs) is critical for understanding virus-host relationships, limitations on the scalability of high-throughput methods have hampered their identification beyond a number of well-studied viruses. Here, we implement an in silico computational framework (pathogen host interactome prediction using structure similarity [P-HIPSTer]) that employs structural information to predict â¼282,000 pan viral-human PPIs with an experimental validation rate of â¼76%. In addition to rediscovering known biology, P-HIPSTer has yielded a series of new findings: the discovery of shared and unique machinery employed across human-infecting viruses, a likely role for ZIKV-ESR1 interactions in modulating viral replication, the identification of PPIs that discriminate between human papilloma viruses (HPVs) with high and low oncogenic potential, and a structure-enabled history of evolutionary selective pressure imposed on the human proteome. Further, P-HIPSTer enables discovery of previously unappreciated cellular circuits that act on human-infecting viruses and provides insight into experimentally intractable viruses.
Asunto(s)
Interacciones Huésped-Patógeno , Mapeo de Interacción de Proteínas , Proteoma/metabolismo , Proteínas Virales/metabolismo , Virus Zika/fisiología , Animales , Atlas como Asunto , Chlorocebus aethiops , Simulación por Computador , Conjuntos de Datos como Asunto , Células HEK293 , Humanos , Células MCF-7 , Proteoma/química , Células Vero , Proteínas Virales/químicaRESUMEN
Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.
Asunto(s)
Anticuerpos/uso terapéutico , Aprendizaje Profundo , Diagnóstico por Computador/métodos , Quimioterapia Asistida por Computador/métodos , Neoplasias/patología , Animales , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones SCID , Metástasis de la Neoplasia , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Programas Informáticos , Microambiente TumoralRESUMEN
Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.
Asunto(s)
Proteína BRCA1/genética , Neoplasias de la Mama/genética , Transdiferenciación Celular/genética , Daño del ADN/genética , Reparación del ADN/genética , Glándulas Mamarias Animales/patología , Animales , Proteína BRCA1/metabolismo , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/patología , Diferenciación Celular/genética , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Femenino , Células HEK293 , Humanos , Células MCF-7 , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción HES-1/metabolismo , TransfecciónRESUMEN
Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.
Asunto(s)
Neoplasias de la Mama/metabolismo , Antagonistas del Receptor de Estrógeno/farmacología , Fulvestrant/farmacología , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Cinamatos/farmacología , Resistencia a Antineoplásicos , Antagonistas del Receptor de Estrógeno/uso terapéutico , Femenino , Fulvestrant/uso terapéutico , Células HEK293 , Xenoinjertos , Humanos , Indazoles/farmacología , Ligandos , Células MCF-7 , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Polimorfismo de Nucleótido Simple , Proteolisis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
CD8+ T cell exhaustion dampens antitumor immunity. Although several transcription factors have been identified that regulate T cell exhaustion, the molecular mechanisms by which CD8+ T cells are triggered to enter an exhausted state remain unclear. Here, we show that interleukin-2 (IL-2) acts as an environmental cue to induce CD8+ T cell exhaustion within tumor microenvironments. We find that a continuously high level of IL-2 leads to the persistent activation of STAT5 in CD8+ T cells, which in turn induces strong expression of tryptophan hydroxylase 1, thus catalyzing the conversion to tryptophan to 5-hydroxytryptophan (5-HTP). 5-HTP subsequently activates AhR nuclear translocation, causing a coordinated upregulation of inhibitory receptors and downregulation of cytokine and effector-molecule production, thereby rendering T cells dysfunctional in the tumor microenvironment. This molecular pathway is not only present in mouse tumor models but is also observed in people with cancer, identifying IL-2 as a novel inducer of T cell exhaustion.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Interleucina-2/metabolismo , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Neoplasias/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Microambiente Tumoral , 5-Hidroxitriptófano/metabolismo , Animales , Anticuerpos Neutralizantes/farmacología , Antineoplásicos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Células HEK293 , Humanos , Interleucina-2/antagonistas & inhibidores , Interleucina-2/genética , Células Jurkat , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Células MCF-7 , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias/patología , Receptores de Hidrocarburo de Aril/deficiencia , Receptores de Hidrocarburo de Aril/genética , Transducción de Señal , Triptófano Hidroxilasa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chromatin regulators play a broad role in regulating gene expression and, when gone awry, can lead to cancer. Here, we demonstrate that ablation of the histone demethylase LSD1 in cancer cells increases repetitive element expression, including endogenous retroviral elements (ERVs), and decreases expression of RNA-induced silencing complex (RISC) components. Significantly, this leads to double-stranded RNA (dsRNA) stress and activation of type 1 interferon, which stimulates anti-tumor T cell immunity and restrains tumor growth. Furthermore, LSD1 depletion enhances tumor immunogenicity and T cell infiltration in poorly immunogenic tumors and elicits significant responses of checkpoint blockade-refractory mouse melanoma to anti-PD-1 therapy. Consistently, TCGA data analysis shows an inverse correlation between LSD1 expression and CD8+ T cell infiltration in various human cancers. Our study identifies LSD1 as a potent inhibitor of anti-tumor immunity and responsiveness to immunotherapy and suggests LSD1 inhibition combined with PD-(L)1 blockade as a novel cancer treatment strategy.
Asunto(s)
Retrovirus Endógenos/genética , Histona Demetilasas/metabolismo , Complejo Silenciador Inducido por ARN/genética , Animales , Línea Celular Tumoral , Cromatina , Terapia Combinada , Regulación de la Expresión Génica/genética , Histona Demetilasas/genética , Humanos , Inmunidad Celular , Inmunoterapia , Interferón Tipo I , Células MCF-7 , Ratones , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , ARN Bicatenario/genética , Linfocitos TRESUMEN
Carcinoma-associated fibroblasts (CAFs) are abundant and heterogeneous stromal cells in tumor microenvironment that are critically involved in cancer progression. Here, we demonstrate that two cell-surface molecules, CD10 and GPR77, specifically define a CAF subset correlated with chemoresistance and poor survival in multiple cohorts of breast and lung cancer patients. CD10+GPR77+ CAFs promote tumor formation and chemoresistance by providing a survival niche for cancer stem cells (CSCs). Mechanistically, CD10+GPR77+ CAFs are driven by persistent NF-κB activation via p65 phosphorylation and acetylation, which is maintained by complement signaling via GPR77, a C5a receptor. Furthermore, CD10+GPR77+ CAFs promote successful engraftment of patient-derived xenografts (PDXs), and targeting these CAFs with a neutralizing anti-GPR77 antibody abolishes tumor formation and restores tumor chemosensitivity. Our study reveals a functional CAF subset that can be defined and isolated by specific cell-surface markers and suggests that targeting the CD10+GPR77+ CAF subset could be an effective therapeutic strategy against CSC-driven solid tumors.
Asunto(s)
Transformación Celular Neoplásica/inmunología , Resistencia a Antineoplásicos/inmunología , Fibroblastos/inmunología , Neoplasias/inmunología , Células Madre Neoplásicas/inmunología , Neprilisina/inmunología , Receptores de Quimiocina/inmunología , Microambiente Tumoral/inmunología , Células A549 , Transformación Celular Neoplásica/patología , Fibroblastos/patología , Humanos , Células MCF-7 , Proteínas de Neoplasias/inmunología , Neoplasias/patología , Células Madre Neoplásicas/patología , Receptor de Anafilatoxina C5aRESUMEN
Approximately half of human genes generate mRNAs with alternative 3' untranslated regions (3'UTRs). Through 3'UTR-mediated protein-protein interactions, alternative 3'UTRs enable multi-functionality of proteins with identical amino acid sequence. While studying how information on protein features is transferred from 3'UTRs to proteins, we discovered that the broadly expressed RNA-binding protein TIS11B forms a membraneless organelle, called TIS granule, that enriches membrane protein-encoding mRNAs with multiple AU-rich elements. TIS granules form a reticular meshwork intertwined with the endoplasmic reticulum (ER). The association between TIS granules and the ER creates a subcellular compartment-the TIGER domain-with a biophysically and biochemically distinct environment from the cytoplasm. This compartment promotes 3'UTR-mediated interaction of SET with membrane proteins, thus allowing increased surface expression and functional diversity of proteins, including CD47 and PD-L1. The TIGER domain is a subcellular compartment that enables formation of specific and functionally relevant protein-protein interactions that cannot be established outside.
Asunto(s)
Regiones no Traducidas 3' , Gránulos Citoplasmáticos/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Factor 1 de Respuesta al Butirato , Antígeno CD47/genética , Antígeno CD47/metabolismo , Gránulos Citoplasmáticos/genética , Drosophila melanogaster , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Células MCF-7 , Ratones , Células 3T3 NIH , Proteínas Nucleares/genética , Dominios Proteicos , Proteínas de Unión al ARN/genéticaRESUMEN
Mammalian folate metabolism is comprised of cytosolic and mitochondrial pathways with nearly identical core reactions, yet the functional advantages of such an organization are not well understood. Using genome-editing and biochemical approaches, we find that ablating folate metabolism in the mitochondria of mammalian cell lines results in folate degradation in the cytosol. Mechanistically, we show that QDPR, an enzyme in tetrahydrobiopterin metabolism, moonlights to repair oxidative damage to tetrahydrofolate (THF). This repair capacity is overwhelmed when cytosolic THF hyperaccumulates in the absence of mitochondrially produced formate, leading to THF degradation. Unexpectedly, we also find that the classic antifolate methotrexate, by inhibiting its well-known target DHFR, causes even more extensive folate degradation in nearly all tested cancer cell lines. These findings shed light on design features of folate metabolism, provide a biochemical basis for clinically observed folate deficiency in QDPR-deficient patients, and reveal a hitherto unknown and unexplored cellular effect of methotrexate.
Asunto(s)
Carbono/metabolismo , Citosol/metabolismo , Formiatos/metabolismo , Mitocondrias/metabolismo , Neoplasias/metabolismo , Tetrahidrofolatos/metabolismo , Citosol/patología , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Metotrexato/farmacocinética , Metotrexato/farmacología , Mitocondrias/patología , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed "multiplexed proteome dynamics profiling" (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteoma/análisis , Proteómica/métodos , Azepinas/química , Azepinas/metabolismo , Azepinas/farmacología , Línea Celular , Cromatografía Líquida de Alta Presión , Análisis por Conglomerados , Estradiol/farmacología , Humanos , Marcaje Isotópico , Células Jurkat , Células MCF-7 , Proteínas de Neoplasias/metabolismo , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Proteolisis/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Espectrometría de Masas en Tándem , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacologíaRESUMEN
Many tumors produce platelet-derived growth factor (PDGF)-DD, which promotes cellular proliferation, epithelial-mesenchymal transition, stromal reaction, and angiogenesis through autocrine and paracrine PDGFRß signaling. By screening a secretome library, we found that the human immunoreceptor NKp44, encoded by NCR2 and expressed on natural killer (NK) cells and innate lymphoid cells, recognizes PDGF-DD. PDGF-DD engagement of NKp44 triggered NK cell secretion of interferon gamma (IFN)-γ and tumor necrosis factor alpha (TNF-α) that induced tumor cell growth arrest. A distinctive transcriptional signature of PDGF-DD-induced cytokines and the downregulation of tumor cell-cycle genes correlated with NCR2 expression and greater survival in glioblastoma. NKp44 expression in mouse NK cells controlled the dissemination of tumors expressing PDGF-DD more effectively than control mice, an effect enhanced by blockade of the inhibitory receptor CD96 or CpG-oligonucleotide treatment. Thus, while cancer cell production of PDGF-DD supports tumor growth and stromal reaction, it concomitantly activates innate immune responses to tumor expansion.
Asunto(s)
Neoplasias Encefálicas/inmunología , Puntos de Control del Ciclo Celular , Glioblastoma/inmunología , Células Asesinas Naturales/inmunología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Neoplasias Encefálicas/patología , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Femenino , Glioblastoma/patología , Humanos , Inmunidad Innata , Interferón gamma/metabolismo , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos C57BL , Receptor 2 Gatillante de la Citotoxidad Natural/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT.
Asunto(s)
Neoplasias de la Mama/patología , Exosomas/patología , ARN no Traducido/metabolismo , Células del Estroma/patología , Microambiente Tumoral , Neoplasias de la Mama/metabolismo , Proteína 58 DEAD Box/metabolismo , Exosomas/metabolismo , Humanos , Factores Reguladores del Interferón/metabolismo , Células MCF-7 , Metástasis de la Neoplasia , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , Receptores Inmunológicos , Receptores de Reconocimiento de Patrones/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Células del Estroma/metabolismo , Virosis/metabolismoRESUMEN
The estrogen receptor (ER), glucocorticoid receptor (GR), and forkhead box protein 1 (FoxA1) are significant factors in breast cancer progression. FoxA1 has been implicated in establishing ER-binding patterns though its unique ability to serve as a pioneer factor. However, the molecular interplay between ER, GR, and FoxA1 requires further investigation. Here we show that ER and GR both have the ability to alter the genomic distribution of the FoxA1 pioneer factor. Single-molecule tracking experiments in live cells reveal a highly dynamic interaction of FoxA1 with chromatin in vivo. Furthermore, the FoxA1 factor is not associated with detectable footprints at its binding sites throughout the genome. These findings support a model wherein interactions between transcription factors and pioneer factors are highly dynamic. Moreover, at a subset of genomic sites, the role of pioneer can be reversed, with the steroid receptors serving to enhance binding of FoxA1.
Asunto(s)
Factor Nuclear 3-alfa del Hepatocito/metabolismo , Cromatina/metabolismo , Desoxirribonucleasas/metabolismo , Humanos , Células MCF-7 , Receptores de Estrógenos/genética , Receptores de Glucocorticoides/genética , Factores de Transcripción/metabolismoRESUMEN
The HIF transcription factor promotes adaptation to hypoxia and stimulates the growth of certain cancers, including triple-negative breast cancer (TNBC). The HIFα subunit is usually prolyl-hydroxylated by EglN family members under normoxic conditions, causing its rapid degradation. We confirmed that TNBC cells secrete glutamate, which we found is both necessary and sufficient for the paracrine induction of HIF1α in such cells under normoxic conditions. Glutamate inhibits the xCT glutamate-cystine antiporter, leading to intracellular cysteine depletion. EglN1, the main HIFα prolyl-hydroxylase, undergoes oxidative self-inactivation in the absence of cysteine both in biochemical assays and in cells, resulting in HIF1α accumulation. Therefore, EglN1 senses both oxygen and cysteine.
Asunto(s)
Neoplasias de la Mama/metabolismo , Cisteína/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Comunicación Paracrina , Neoplasias de la Mama Triple Negativas/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Animales , Ácido Glutámico/metabolismo , Humanos , Células MCF-7 , RatonesRESUMEN
Immune cells identify and destroy tumors by recognizing cellular traits indicative of oncogenic transformation. In this study, we found that myocardin-related transcription factors (MRTFs), which promote migration and metastatic invasion, also sensitize cancer cells to the immune system. Melanoma and breast cancer cells with high MRTF expression were selectively eliminated by cytotoxic lymphocytes in mouse models of metastasis. This immunosurveillance phenotype was further enhanced by treatment with immune checkpoint blockade (ICB) antibodies. We also observed that high MRTF signaling in human melanoma is associated with ICB efficacy in patients. Using biophysical and functional assays, we showed that MRTF overexpression rigidified the filamentous actin cytoskeleton and that this mechanical change rendered mouse and human cancer cells more vulnerable to cytotoxic T lymphocytes and natural killer cells. Collectively, these results suggest that immunosurveillance has a mechanical dimension, which we call mechanosurveillance, that is particularly relevant for the targeting of metastatic disease.
Asunto(s)
Linfocitos/inmunología , Neoplasias/inmunología , Citoesqueleto de Actina/inmunología , Actinas/inmunología , Animales , Comunicación Celular/inmunología , Línea Celular , Línea Celular Tumoral , Movimiento Celular/inmunología , Femenino , Células HEK293 , Humanos , Células Asesinas Naturales/inmunología , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/inmunología , Factores de Transcripción/inmunologíaRESUMEN
Stromal cells within the tumor microenvironment are essential for tumor progression and metastasis. Surprisingly little is known about the factors that drive the transcriptional reprogramming of stromal cells within tumors. We report that the transcriptional regulator heat shock factor 1 (HSF1) is frequently activated in cancer-associated fibroblasts (CAFs), where it is a potent enabler of malignancy. HSF1 drives a transcriptional program in CAFs that complements, yet is completely different from, the program it drives in adjacent cancer cells. This CAF program is uniquely structured to support malignancy in a non-cell-autonomous way. Two central stromal signaling molecules-TGF-ß and SDF1-play a critical role. In early-stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. Thus, tumors co-opt the ancient survival functions of HSF1 to orchestrate malignancy in both cell-autonomous and non-cell-autonomous ways, with far-reaching therapeutic implications.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Pulmonares/metabolismo , Factores de Transcripción/metabolismo , Animales , Quimiocina CXCL12/metabolismo , Fibroblastos/metabolismo , Factores de Transcripción del Choque Térmico , Xenoinjertos , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Adenosine-to-inosine editing is catalyzed by ADAR1 at thousands of sites transcriptome-wide. Despite intense interest in ADAR1 from physiological, bioengineering, and therapeutic perspectives, the rules of ADAR1 substrate selection are poorly understood. Here, we used large-scale systematic probing of â¼2,000 synthetic constructs to explore the structure and sequence context determining editability. We uncover two structural layers determining the formation and propagation of A-to-I editing, independent of sequence. First, editing is robustly induced at fixed intervals of 35 bp upstream and 30 bp downstream of structural disruptions. Second, editing is symmetrically introduced on opposite sites on a double-stranded structure. Our findings suggest a recursive model for RNA editing, whereby the structural alteration induced by the editing at one site iteratively gives rise to the formation of an additional editing site at a fixed periodicity, serving as a basis for the propagation of editing along and across both strands of double-stranded RNA structures.
Asunto(s)
Adenosina Desaminasa/genética , Adenosina/metabolismo , Inosina/metabolismo , Edición de ARN , ARN Bicatenario/genética , Proteínas de Unión al ARN/genética , Células A549 , Adenosina/genética , Adenosina Desaminasa/metabolismo , Animales , Emparejamiento Base , Células HEK293 , Humanos , Inosina/genética , Células MCF-7 , Ratones , Células 3T3 NIH , Conformación de Ácido Nucleico , ARN Bicatenario/química , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
The transition of oxidized 5-methylcytosine (5mC) intermediates into the base excision repair (BER) pipeline to complete DNA demethylation remains enigmatic. We report here that UHRF2, the only paralog of UHRF1 in mammals that fails to rescue Uhrf1-/- phenotype, is physically and functionally associated with BER complex. We show that UHRF2 is allosterically activated by 5-hydroxymethylcytosine (5hmC) and acts as a ubiquitin E3 ligase to catalyze K33-linked polyubiquitination of XRCC1. This nonproteolytic action stimulates XRCC1's interaction with the ubiquitin binding domain-bearing RAD23B, leading to the incorporation of TDG into BER complex. Integrative epigenomic analysis in mouse embryonic stem cells reveals that Uhrf2-fostered TDG-RAD23B-BER complex is functionally linked to the completion of DNA demethylation at active promoters and that Uhrf2 ablation impedes DNA demethylation on latent enhancers that undergo poised-to-active transition during neuronal commitment. Together, these observations highlight an essentiality of 5hmC-switched UHRF2 E3 ligase activity in commissioning the accomplishment of active DNA demethylation.