RESUMEN
The immune microenvironment of hepatocellular carcinoma (HCC) is poorly characterized. Combining two single-cell RNA sequencing technologies, we produced transcriptomes of CD45+ immune cells for HCC patients from five immune-relevant sites: tumor, adjacent liver, hepatic lymph node (LN), blood, and ascites. A cluster of LAMP3+ dendritic cells (DCs) appeared to be the mature form of conventional DCs and possessed the potential to migrate from tumors to LNs. LAMP3+ DCs also expressed diverse immune-relevant ligands and exhibited potential to regulate multiple subtypes of lymphocytes. Of the macrophages in tumors that exhibited distinct transcriptional states, tumor-associated macrophages (TAMs) were associated with poor prognosis, and we established the inflammatory role of SLC40A1 and GPNMB in these cells. Further, myeloid and lymphoid cells in ascites were predominantly linked to tumor and blood origins, respectively. The dynamic properties of diverse CD45+ cell types revealed by this study add new dimensions to the immune landscape of HCC.
Asunto(s)
Carcinoma Hepatocelular/inmunología , Proteínas de Transporte de Catión/genética , Inflamación/inmunología , Neoplasias Hepáticas/inmunología , Glicoproteínas de Membrana/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Comunicación Celular/genética , Comunicación Celular/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Células Dendríticas/inmunología , Células Dendríticas/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/patología , Antígenos Comunes de Leucocito/inmunología , Hígado/inmunología , Hígado/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Linfocitos/inmunología , Linfocitos/patología , Proteínas de Membrana de los Lisosomas/genética , Macrófagos/inmunología , Macrófagos/patología , Células Mieloides/inmunología , Células Mieloides/patología , Proteínas de Neoplasias/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transcriptoma/genética , Transcriptoma/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Linfocitos T CD8-positivos/fisiología , Factor de Transcripción AP-1/metabolismo , Virus Vaccinia/inmunología , Vaccinia/inmunología , Inmunidad Adaptativa , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Células Cultivadas , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Memoria Inmunológica/genética , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Oncogénica p65(gag-jun) , Transducción de Señal/genética , Factor de Transcripción AP-1/genéticaRESUMEN
Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.
Asunto(s)
Genes Esenciales/genética , Inmunoterapia , Neoplasias/genética , Neoplasias/terapia , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Presentación de Antígeno/genética , Apelina/metabolismo , Receptores de Apelina/genética , Receptores de Apelina/metabolismo , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Femenino , Genoma/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Interferón gamma/inmunología , Janus Quinasa 1/metabolismo , Bases del Conocimiento , Melanoma/genética , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/terapia , Ratones , Mutación , Neoplasias/inmunología , Neoplasias/metabolismo , Reproducibilidad de los Resultados , Linfocitos T Citotóxicos/metabolismoRESUMEN
Tumours progress despite being infiltrated by tumour-specific effector T cells. Tumours contain areas of cellular necrosis, which are associated with poor survival in a variety of cancers. Here, we show that necrosis releases intracellular potassium ions into the extracellular fluid of mouse and human tumours, causing profound suppression of T cell effector function. Elevation of the extracellular potassium concentration ([K+]e) impairs T cell receptor (TCR)-driven Akt-mTOR phosphorylation and effector programmes. Potassium-mediated suppression of Akt-mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A. Although the suppressive effect mediated by elevated [K+]e is independent of changes in plasma membrane potential (Vm), it requires an increase in intracellular potassium ([K+]i). Accordingly, augmenting potassium efflux in tumour-specific T cells by overexpressing the potassium channel Kv1.3 lowers [K+]i and improves effector functions in vitro and in vivo and enhances tumour clearance and survival in melanoma-bearing mice. These results uncover an ionic checkpoint that blocks T cell function in tumours and identify potential new strategies for cancer immunotherapy.
Asunto(s)
Cationes Monovalentes/metabolismo , Melanoma/inmunología , Potasio/metabolismo , Linfocitos T/inmunología , Escape del Tumor/inmunología , Microambiente Tumoral/inmunología , Animales , Humanos , Tolerancia Inmunológica/inmunología , Inmunoterapia/métodos , Canal de Potasio Kv1.3/metabolismo , Masculino , Melanoma/metabolismo , Melanoma/patología , Melanoma/terapia , Potenciales de la Membrana , Ratones , Necrosis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Análisis de Supervivencia , Linfocitos T/metabolismo , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Although prostate cancer typically runs an indolent course, a subset of men develop aggressive, fatal forms of this disease. We hypothesize that germline variation modulates susceptibility to aggressive prostate cancer. The goal of this work is to identify susceptibility genes using the C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of neuroendocrine prostate cancer. Quantitative trait locus (QTL) mapping was performed in transgene-positive (TRAMPxNOD/ShiLtJ) F2 intercross males (nâ=â228), which facilitated identification of 11 loci associated with aggressive disease development. Microarray data derived from 126 (TRAMPxNOD/ShiLtJ) F2 primary tumors were used to prioritize candidate genes within QTLs, with candidate genes deemed as being high priority when possessing both high levels of expression-trait correlation and a proximal expression QTL. This process enabled the identification of 35 aggressive prostate tumorigenesis candidate genes. The role of these genes in aggressive forms of human prostate cancer was investigated using two concurrent approaches. First, logistic regression analysis in two human prostate gene expression datasets revealed that expression levels of five genes (CXCL14, ITGAX, LPCAT2, RNASEH2A, and ZNF322) were positively correlated with aggressive prostate cancer and two genes (CCL19 and HIST1H1A) were protective for aggressive prostate cancer. Higher than average levels of expression of the five genes that were positively correlated with aggressive disease were consistently associated with patient outcome in both human prostate cancer tumor gene expression datasets. Second, three of these five genes (CXCL14, ITGAX, and LPCAT2) harbored polymorphisms associated with aggressive disease development in a human GWAS cohort consisting of 1,172 prostate cancer patients. This study is the first example of using a systems genetics approach to successfully identify novel susceptibility genes for aggressive prostate cancer. Such approaches will facilitate the identification of novel germline factors driving aggressive disease susceptibility and allow for new insights into these deadly forms of prostate cancer.
Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa/genética , Antígeno CD11c/genética , Quimiocinas CXC/genética , Neoplasias de la Próstata/genética , Animales , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Ratones , Neoplasias de la Próstata/patología , Sitios de Carácter Cuantitativo/genética , Ribonucleasa H/genéticaRESUMEN
Effective eradication of leukemic stem cells (LSCs) remains the greatest challenge in treating acute myeloid leukemia (AML). The immune receptor LAIR-1 has been shown to regulate LSC survival; however, the therapeutic potential of this pathway remains unexplored. We developed a therapeutic LAIR-1 agonist antibody, NC525, that induced cell death of LSCs, but not healthy hematopoietic stem cells in vitro, and killed LSCs and AML blasts in both cell- and patient-derived xenograft models. We showed that LAIR-1 agonism drives a unique apoptotic signaling program in leukemic cells that was enhanced in the presence of collagen. NC525 also significantly improved the activity of azacitidine and venetoclax to establish LAIR-1 targeting as a therapeutic strategy for AML that may synergize with standard-of-care therapies.
Asunto(s)
Leucemia Mieloide Aguda , Animales , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Células Madre Hematopoyéticas/metabolismo , Transducción de Señal , Modelos Animales de Enfermedad , Células Madre Neoplásicas/metabolismoRESUMEN
Adoptive T cell therapies (ACT) have been curative for a limited number of cancer patients. The sensitization of cancer cells to T cell killing may expand the benefit of these therapies for more patients. To this end, we use a three-step approach to identify cancer genes that disfavor T cell immunity. First, we profile gene transcripts upregulated by cancer under selection pressure from T cell killing. Second, we identify potential tumor gene targets and pathways that disfavor T cell killing using signaling pathway activation libraries and genome-wide loss-of-function CRISPR-Cas9 screens. Finally, we implement pharmacological perturbation screens to validate these targets and identify BIRC2, ITGAV, DNPEP, BCL2, and ERRα as potential ACT-drug combination candidates. Here, we establish that BIRC2 limits antigen presentation and T cell recognition of tumor cells by suppressing IRF1 activity and provide evidence that BIRC2 inhibition in combination with ACT is an effective strategy to increase efficacy.
Asunto(s)
Neoplasias , Linfocitos T , Presentación de Antígeno , Sistemas CRISPR-Cas/genética , Humanos , Neoplasias/genética , Oncogenes , Análisis de SistemasRESUMEN
BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. METHODS: Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. FINDINGS: CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo. CONCLUSIONS: CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. FUNDING: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.
Asunto(s)
Linfocitos Infiltrantes de Tumor , Linfocitos T , Traslado Adoptivo , Animales , Citocinas/metabolismo , Humanos , Inmunoterapia Adoptiva/métodos , RatonesRESUMEN
T cells are central to all currently effective cancer immunotherapies, but the characteristics defining therapeutically effective anti-tumor T cells have not been comprehensively elucidated. Here, we delineate four phenotypic qualities of effective anti-tumor T cells: cell expansion, differentiation, oxidative stress, and genomic stress. Using a CRISPR-Cas9-based genetic screen of primary T cells we measured the multi-phenotypic impact of disrupting 25 T cell receptor-driven kinases. We identified p38 kinase as a central regulator of all four phenotypes and uncovered transcriptional and antioxidant pathways regulated by p38 in T cells. Pharmacological inhibition of p38 improved the efficacy of mouse anti-tumor T cells and enhanced the functionalities of human tumor-reactive and gene-engineered T cells, paving the way for clinically relevant interventions.
Asunto(s)
Neoplasias de la Mama/terapia , Sistemas CRISPR-Cas , Inmunoterapia Adoptiva/métodos , Melanoma Experimental/terapia , Fenotipo , Linfocitos T/trasplante , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Diferenciación Celular , Femenino , Ingeniería Genética , Masculino , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
A paradox of tumor immunology is that tumor-infiltrating lymphocytes are dysfunctional in situ, yet are capable of stem cell-like behavior including self-renewal, expansion, and multipotency, resulting in the eradication of large metastatic tumors. We find that the overabundance of potassium in the tumor microenvironment underlies this dichotomy, triggering suppression of T cell effector function while preserving stemness. High levels of extracellular potassium constrain T cell effector programs by limiting nutrient uptake, thereby inducing autophagy and reduction of histone acetylation at effector and exhaustion loci, which in turn produces CD8+ T cells with improved in vivo persistence, multipotency, and tumor clearance. This mechanistic knowledge advances our understanding of T cell dysfunction and may lead to novel approaches that enable the development of enhanced T cell strategies for cancer immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Tolerancia Inmunológica , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias/inmunología , Potasio/metabolismo , Células Madre/inmunología , Acetilcoenzima A/metabolismo , Acetilación , Animales , Autofagia/inmunología , Restricción Calórica , Diferenciación Celular/genética , Epigénesis Genética , Histonas/metabolismo , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Microambiente TumoralRESUMEN
To better elucidate epigenetic mechanisms that correlate with the dynamic gene expression program observed upon T-cell differentiation, we investigated the genomic landscape of histone modifications in naive and memory CD8(+) T cells. Using a ChIP-Seq approach coupled with global gene expression profiling, we generated genome-wide histone H3 lysine 4 (H3K4me3) and H3 lysine 27 (H3K27me3) trimethylation maps in naive, T memory stem cells, central memory cells, and effector memory cells in order to gain insight into how histone architecture is remodeled during T cell differentiation. We show that H3K4me3 histone modifications are associated with activation of genes, while H3K27me3 is negatively correlated with gene expression at canonical loci and enhancers associated with T-cell metabolism, effector function, and memory. Our results also reveal histone modifications and gene expression signatures that distinguish the recently identified T memory stem cells from other CD8(+) T-cell subsets. Taken together, our results suggest that CD8(+) lymphocytes undergo chromatin remodeling in a progressive fashion. These findings have major implications for our understanding of peripheral T-cell ontogeny and the formation of immunological memory.
Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Epigénesis Genética , Animales , Ensamble y Desensamble de Cromatina/genética , Elementos de Facilitación Genéticos/genética , Perfilación de la Expresión Génica , Histonas/metabolismo , Memoria Inmunológica/genética , Subgrupos Linfocitarios/metabolismo , Metilación , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-PostraduccionalRESUMEN
Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.
Asunto(s)
Células Progenitoras Linfoides/fisiología , Melanoma Experimental/terapia , Potencial de la Membrana Mitocondrial , Subgrupos de Linfocitos T/fisiología , Animales , Linfocitos T CD8-positivos/fisiología , Línea Celular Tumoral , Citocinas/fisiología , Células Madre Hematopoyéticas/fisiología , Humanos , Células Progenitoras Linfoides/trasplante , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante de Neoplasias , Estrés Oxidativo , Trasplante de Células Madre , Subgrupos de Linfocitos T/trasplante , TranscriptomaRESUMEN
The immune system has a powerful ability to recognize and kill cancer cells, but its function is often suppressed within tumors, preventing clearance of disease. Functionally diverse innate and adaptive cellular lineages either drive or constrain immune reactions within tumors. The transcription factor (TF) BACH2 regulates the differentiation of multiple innate and adaptive cellular lineages, but its role in controlling tumor immunity has not been elucidated. Here, we demonstrate that BACH2 is required to establish immunosuppression within tumors. Tumor growth was markedly impaired in Bach2-deficient mice and coincided with intratumoral activation of both innate and adaptive immunity. However, augmented tumor clearance in the absence of Bach2 was dependent upon the adaptive immune system. Analysis of tumor-infiltrating lymphocytes from Bach2-deficient mice revealed high frequencies of rapidly proliferating effector CD4+ and CD8+ T cells that expressed the inflammatory cytokine IFN-γ. Effector T cell activation coincided with a reduction in the frequency of intratumoral Foxp3+ Tregs. Mechanistically, BACH2 promoted tumor immunosuppression through Treg-mediated inhibition of intratumoral CD8+ T cells and IFN-γ. These findings demonstrate that BACH2 is a key component of the molecular program of tumor immunosuppression and identify therapeutic targets for the reversal of immunosuppression in cancer.
Asunto(s)
Inmunidad Adaptativa , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Linfocitos T CD8-positivos/inmunología , Inmunidad Innata , Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Linfocitos T CD8-positivos/patología , Interferón gamma/genética , Interferón gamma/inmunología , Ratones , Ratones Noqueados , Neoplasias/genética , Neoplasias/patología , Linfocitos T Reguladores/patologíaRESUMEN
Prostate cancer (PC) is very common in developed countries. However, the molecular determinants of PC metastasis are unclear. Previously, we reported that germline variation influences metastasis in the C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of PC. These mice develop prostate tumors similar to a subset of poor outcome, treatment-associated human PC tumors. Here, we used TRAMP mice to nominate candidate genes and validate their role in aggressive human PC in PC datasets and cell lines. Candidate metastasis susceptibility genes were identified through quantitative trait locus (QTL) mapping in 201 (TRAMP × PWK/PhJ) F2 males. Two metastasis-associated QTLs were identified; one on chromosome 12 (LOD = 5.86), and one on chromosome 14 (LOD = 4.41). Correlation analysis using microarray data from (TRAMP × PWK/PhJ) F2 prostate tumors identified 35 metastasis-associated transcripts within the two loci. The role of these genes in susceptibility to aggressive human PC was determined through in silico analysis using multiple datasets. First, analysis of candidate gene expression in two human PC datasets demonstrated that five candidate genes were associated with an increased risk of aggressive disease and lower disease-free survival. Second, four of these genes (GNL3, MAT1A, SKA3, and ZMYM5) harbored SNPs associated with aggressive tumorigenesis in the PLCO/CGEMS GWAS of 1172 PC patients. Finally, over-expression of GNL3 and SKA3 in the PC-3 human PC cell line decreased in vitro cell migration and invasion. This novel approach demonstrates how mouse models can be used to identify metastasis susceptibility genes, and gives new insight into the molecular mechanisms of fatal PC.
Asunto(s)
Proteínas de Unión al GTP/genética , Predisposición Genética a la Enfermedad , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Neoplasias de la Próstata/genética , Animales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Modelos Animales de Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Metástasis de la Neoplasia , Polimorfismo de Nucleótido Simple , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/patología , Sitios de Carácter CuantitativoRESUMEN
Improving the functional avidity of effector T cells is critical in overcoming inhibitory factors within the tumor microenvironment and eliciting tumor regression. We have found that Cish, a member of the suppressor of cytokine signaling (SOCS) family, is induced by TCR stimulation in CD8(+) T cells and inhibits their functional avidity against tumors. Genetic deletion of Cish in CD8(+) T cells enhances their expansion, functional avidity, and cytokine polyfunctionality, resulting in pronounced and durable regression of established tumors. Although Cish is commonly thought to block STAT5 activation, we found that the primary molecular basis of Cish suppression is through inhibition of TCR signaling. Cish physically interacts with the TCR intermediate PLC-γ1, targeting it for proteasomal degradation after TCR stimulation. These findings establish a novel targetable interaction that regulates the functional avidity of tumor-specific CD8(+) T cells and can be manipulated to improve adoptive cancer immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Melanoma Experimental/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Proteínas Supresoras de la Señalización de Citocinas/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Células Cultivadas , Humanos , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/inmunología , Immunoblotting , Inmunoterapia Adoptiva/métodos , Melanoma Experimental/genética , Melanoma Experimental/terapia , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfolipasa C gamma/inmunología , Fosfolipasa C gamma/metabolismo , Unión Proteica/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de la Señalización de Citocinas/deficiencia , Proteínas Supresoras de la Señalización de Citocinas/genética , Transcriptoma/genética , Transcriptoma/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
UNLABELLED: Overexpression of ribosomal RNA processing 1 homolog B (RRP1B) induces a transcriptional profile that accurately predicts patient outcome in breast cancer. However, the mechanism by which RRP1B modulates transcription is unclear. Here, the chromatin-binding properties of RRP1B were examined to define how it regulates metastasis-associated transcription. To identify genome-wide RRP1B-binding sites, high-throughput ChIP-seq was performed in the human breast cancer cell line MDA-MB-231 and HeLa cells using antibodies against endogenous RRP1B. Global changes in repressive marks such as histone H3 lysine 9 trimethylation (H3K9me3) were also examined by ChIP-seq. Analysis of these samples identified 339 binding regions in MDA-MB-231 cells and 689 RRP1B-binding regions in HeLa cells. Among these, 136 regions were common to both cell lines. Gene expression analyses of these RRP1B-binding regions revealed that transcriptional repression is the primary result of RRP1B binding to chromatin. ChIP-reChIP assays demonstrated that RRP1B co-occupies loci with decreased gene expression with the heterochromatin-associated proteins, tripartite motif-containing protein 28 (TRIM28/KAP1), and heterochromatin protein 1-α (CBX5/HP1α). RRP1B occupancy at these loci was also associated with higher H3K9me3 levels, indicative of heterochromatinization mediated by the TRIM28/HP1α complex. In addition, RRP1B upregulation, which is associated with metastasis suppression, induced global changes in histone methylation. IMPLICATIONS: RRP1B, a breast cancer metastasis suppressor, regulates gene expression through heterochromatinization and transcriptional repression, which helps our understanding of mechanisms that drive prognostic gene expression in human breast cancer.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias de la Mama/patología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Proteínas Represoras/genética , Animales , Proteínas Reguladoras de la Apoptosis/química , Sitios de Unión , Neoplasias de la Mama/genética , Línea Celular Tumoral , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/química , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células HeLa , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/patología , Metilación , Ratones , Neoplasias Experimentales , Transcripción Genética , Proteína 28 que Contiene Motivos TripartitoRESUMEN
Neuroendocrine (NE) differentiation has gained increased attention as a prostate cancer (PC) prognostic marker. The aim of this study is to determine whether host germline genetic variation influences tumor progression and metastasis in C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of aggressive NEPC. TRAMP mice were crossed to the eight progenitor strains of the Collaborative Cross recombinant inbred panel to address this. Tumor growth and metastasis burden were quantified in heterozygous transgene positive F1 male mice at 30 weeks of age. Compared to wild-type C57BL/6J-Tg(TRAMP)824Ng/J males, TRAMP x CAST/EiJ, TRAMP x NOD/ShiLtJ and TRAMP x NZO/HlLtJ F1 males displayed significant increases in tumor growth. Conversely, TRAMP x WSB/EiJ and TRAMP x PWK/PhJ F1 males displayed significant reductions in tumor growth. Interestingly, despite reduced tumor burden, TRAMP x WSB/EiJ males had an increased nodal metastasis burden. Patterns of distant pulmonary metastasis tended to follow the same patterns as that of local dissemination in each of the strains. All tumors and metastases displayed positive staining for NE markers, synaptophysin, and FOXA2. These experiments conclusively demonstrate that the introduction of germline variation by breeding modulates tumor growth, local metastasis burden, and distant metastasis frequency in this model of NEPC. These strains will be useful as model systems to facilitate the identification of germline modifier genes that promote the development of aggressive forms of PC.