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Patients with ER-negative breast cancer have the worst prognosis of all breast cancer subtypes, often experiencing rapid recurrence or progression to metastatic disease shortly after diagnosis. Given that metastasis is the primary cause of mortality in most solid tumors, understanding metastatic biology is crucial for effective intervention. Using a mouse systems genetics approach, we previously identified 12 genes associated with metastatic susceptibility. Here, we extend those studies to identify Resf1, a poorly characterized gene, as a novel metastasis susceptibility gene in ER- breast cancer. Resf1 is a large, unstructured protein with an evolutionarily conserved intron-exon structure, but with poor amino acid conservation. CRISPR or gene trap mouse models crossed to the Polyoma Middle-T antigen genetically engineered mouse model (MMTV-PyMT) demonstrated that reduction of Resf1 resulted in a significant increase in tumor growth, a shortened overall survival time, and increased incidence and number of lung metastases, consistent with patient data. Furthermore, an analysis of matched tail and primary tissues revealed loss of the wildtype copy in tumor tissue, consistent with Resf1 being a tumor suppressor. Mechanistic analysis revealed a potential role of Resf1 in transcriptional control through association with compound G4 quadruplexes in expressed sequences, particularly those associated with ribosomal biogenesis. These results suggest that loss of Resf1 enhances tumor progression in ER- breast cancer through multiple alterations in both transcriptional and translational control.
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Proteínas Represoras , Neoplasias de la Mama Triple Negativas , Animales , Femenino , Humanos , Ratones , Línea Celular Tumoral , G-Cuádruplex , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/patología , Metástasis de la Neoplasia , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Acetylation of protein and RNA represent a critical event for development and cancer progression. NAT10 is the only known RNA acetylase that catalyzes the N4-actylcytidine (ac4C) modification of RNAs. Here, we show that the loss of NAT10 significantly decreases lung metastasis in allograft and genetically engineered mouse models of breast cancer. NAT10 interacts with a mechanosensitive, metastasis susceptibility protein complex at the nuclear pore. In addition to its canonical role in RNA acetylation, we find that NAT10 interacts with p300 at gene enhancers. NAT10 loss is associated with p300 mislocalization into heterochromatin regions. NAT10 depletion disrupts enhancer organization, leading to alteration of gene transcription necessary for metastatic progression, including reduced myeloid cell-recruiting chemokines that results in a less metastasis-prone tumor microenvironment. Our study uncovers a distinct role of NAT10 in enhancer organization of metastatic tumor cells and suggests its involvement in the tumor-immune crosstalk dictating metastatic outcomes.
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Background: Thyroid cancer cell lines have been of great value for the study of thyroid cancer. However, the availability of benign thyroid adenoma cell lines is limited. Methods: Cell lines were established from thyroid adenomatous nodules that developed in mice treated with the goitrogen amitrole. Expression of epithelial, mesenchymal, and thyroid markers of these established cell lines was determined, and the effect of lentivirus-transduced overexpression of NKX2-1, a master regulator of thyroid development, on the thyroid marker expression was examined. Signal transduction and cell proliferation were evaluated after treatment with insulin-like growth factor-I (IGF-I) and the selective IGF-I receptor (IGF-IR) inhibitor NVP-ADW742. Xenograft studies were performed to examine tumorigenicity of the cells in mice. Whole-genome sequencing (WGS) was used to comprehensively determine the genetic mutations in the established two cell lines. Results: Five mouse thyroid adenomatous nodules-derived cell lines named CAT (cells from amitrole-treated thyroids) were established. Among these, two cell lines, CAT458/458s (CAT458s: a subline of CAT458) and CAT459, were found to be positive for epithelial markers and negative for a mesenchymal marker. NKX2-1-positive CAT459 cells showed higher messenger RNA (mRNA) expression of some thyroid differentiation markers than NKX2-1-negative CAT458s cells, and NKX2-1 overexpression increased and/or induced their expression. IGF-I signaling was transduced in thyrotropin receptor (Tshr)-negative CAT458s and 459 cells, and NVP-ADW742 suppressed their proliferation. No tumors developed in mice after subcutaneous injection of CAT458s or 459 cells. The WGS analysis revealed the presence of missense mutations in the tumor suppressor genes such as Polk (encoding DNA polymerase kappa) and Tgfb1 (encoding transforming growth factor beta 1), while no mutations were found in the prominent thyroid cancer-related genes Braf, Trp53 (encoding p53), and Tert (encoding telomerase reverse transcriptase). Conclusions: Two mouse thyroid adenomatous nodule-derived cell lines with different thyroid differentiation marker expression were established. NKX2-1 induced partial differentiation of these cell lines. They lacked tumorigenicity and prominent gene mutations involved in thyroid cancer development, while missense mutations were found in some tumor suppressors as revealed by WGS. The CAT458s and 459 provide a new tool to further clarify the process of thyroid multistep carcinogenesis and differentiation.
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Factor I del Crecimiento Similar a la Insulina , Neoplasias de la Tiroides , Humanos , Animales , Ratones , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Amitrol (Herbicida) , Neoplasias de la Tiroides/genética , Línea Celular , Línea Celular Tumoral , ADN Polimerasa Dirigida por ADNRESUMEN
BACKGROUND: The absence of prominent, actionable genetic alternations in osteosarcomas (OS) implies that transcriptional and epigenetic mechanisms significantly contribute to the progression of this life-threatening form of cancer. Therefore, the identification of potential transcriptional events that promote the survival of OS cells could be key in devising targeted therapeutic approaches for OS. We have previously shown that RUNX2 is a transcription factor (TF) essential for OS cell survival. Unfortunately, the transcriptional network or circuitry regulated by RUNX2 in OS cells is still largely unknown. METHODS: The TFs that are in the RUNX2 transcriptional circuitry were identified by analyzing RNAseq and ChIPseq datasets of RUNX2. To evaluate the effect of SOX9 knockdown on the survival of osteosarcoma cells in vitro, we employed cleaved caspase-3 immunoblotting and propidium iodide staining techniques. The impact of SOX9 and JMJD1C depletion on OS tumor growth was examined in vivo using xenografts and immunohistochemistry. Downstream targets of SOX9 were identified and dissected using RNAseq, pathway analysis, and gene set enrichment analysis. Furthermore, the interactome of SOX9 was identified using BioID and validated by PLA. RESULT: Our findings demonstrate that SOX9 is a critical TF that is induced by RUNX2. Both in vitro and in vivo experiments revealed that SOX9 plays a pivotal role in the survival of OS. RNAseq analysis revealed that SOX9 activates the transcription of MYC, a downstream target of RUNX2. Mechanistically, our results suggest a transcriptional network involving SOX9, RUNX2, and MYC, with SOX9 binding to RUNX2. Moreover, we discovered that JMJD1C, a chromatin factor, is a novel binding partner of SOX9, and depletion of JMJD1C impairs OS tumor growth. CONCLUSION: The findings of this study represent a significant advancement in our understanding of the transcriptional network present in OS cells, providing valuable insights that may contribute to the development of targeted therapies for OS.
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Intratumoral heterogeneity (ITH) can promote cancer progression and treatment failure, but the complexity of the regulatory programs and contextual factors involved complicates its study. To understand the specific contribution of ITH to immune checkpoint blockade (ICB) response, we generated single cell-derived clonal sublines from an ICB-sensitive and genetically and phenotypically heterogeneous mouse melanoma model, M4. Genomic and single cell transcriptomic analyses uncovered the diversity of the sublines and evidenced their plasticity. Moreover, a wide range of tumor growth kinetics were observed in vivo , in part associated with mutational profiles and dependent on T cell-response. Further inquiry into melanoma differentiation states and tumor microenvironment (TME) subtypes of untreated tumors from the clonal sublines demonstrated correlations between highly inflamed and differentiated phenotypes with the response to anti-CTLA-4 treatment. Our results demonstrate that M4 sublines generate intratumoral heterogeneity at both levels of intrinsic differentiation status and extrinsic TME profiles, thereby impacting tumor evolution during therapeutic treatment. These clonal sublines proved to be a valuable resource to study the complex determinants of response to ICB, and specifically the role of melanoma plasticity in immune evasion mechanisms.
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Secretoglobin (SCGB) 3A2 is a bioactive molecule exhibiting various functions such as improving allergic airway inflammation and pulmonary fibrosis and promoting bronchial branching and proliferation during lung development. To determine if and how SCGB3A2 is involved in chronic obstructive pulmonary disease (COPD), a multifactorial disease with both airway and emphysematous lesions, a COPD mouse model was created by exposing Scgb3a2-deficient (KO), Scgb3a2-lung-specific overexpressing (TG), and wild type (WT) mice to cigarette smoke (CS) for 6 months. The KO mice showed loss of lung structure under control condition, and CS exposure resulted in more expansion of airspace and destruction of alveolar wall than WT mouse lungs. In contrast, TG mouse lungs showed no significant changes after CS exposure. SCGB3A2 increased the expression and phosphorylation of signal transducers and activators of transcription (STAT)1 and STAT3, and the expression of α1-antitrypsin (A1AT) in mouse lung fibroblast-derived MLg cells and mouse lung epithelial-derived MLE-15 cells. In MLg cells, A1AT expression was decreased in Stat3-knockdown cells, and increased upon Stat3 overexpression. STAT3 formed a homodimer when cells were stimulated with SCGB3A2. Chromatin immunoprecipitation and reporter assays demonstrated that STAT3 binds to specific binding sites on the Serpina1a gene encoding A1AT and upregulates its transcription in lung tissues of mice. Furthermore, nuclear localization of phosphorylated STAT3 upon SCGB3A2 stimulation was detected by immunocytochemistry. These findings demonstrate that SCGB3A2 protects the lungs from the development of CS-induced emphysema by regulating A1AT expression through STAT3 signaling.
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Fumar Cigarrillos , Enfermedad Pulmonar Obstructiva Crónica , Enfisema Pulmonar , Fibrosis Pulmonar , Ratones , Animales , Secretoglobinas/genética , Secretoglobinas/metabolismo , Enfisema Pulmonar/genética , Enfisema Pulmonar/prevención & control , Fumar Cigarrillos/efectos adversos , Pulmón/patología , Fibrosis Pulmonar/metabolismo , Inflamación/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismoRESUMEN
Tumor-derived exosomes have documented roles in accelerating the initiation and outgrowth of metastases, as well as in therapy resistance. Little information supports the converse, that exosomes or similar vesicles can suppress metastasis. We investigated the NME1 (Nm23-H1) metastasis suppressor as a candidate for metastasis suppression by extracellular vesicles. Exosomes derived from two cancer cell lines (MDA-MB-231T and MDA-MB-435), when transfected with the NME1 (Nm23-H1) metastasis suppressor, secreted exosomes with NME1 as the predominant constituent. These exosomes entered recipient tumor cells, altered their endocytic patterns in agreement with NME1 function, and suppressed in vitro tumor cell motility and migration compared to exosomes from control transfectants. Proteomic analysis of exosomes revealed multiple differentially expressed proteins that could exert biological functions. Therefore, we also prepared and investigated liposomes, empty or containing partially purified rNME1. rNME1 containing liposomes recapitulated the effects of exosomes from NME1 transfectants in vitro. In an experimental lung metastasis assay the median lung metastases per histologic section was 158 using control liposomes and 15 in the rNME1 liposome group, 90.5% lower than the control liposome group (P = 0.016). The data expand the exosome/liposome field to include metastasis suppressive functions and describe a new translational approach to prevent metastasis.
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Neoplasias de la Mama , Exosomas , Neoplasias Pulmonares , Nucleósido Difosfato Quinasas NM23 , Línea Celular Tumoral , Femenino , Humanos , Liposomas , Neoplasias Pulmonares/secundario , Nucleósido Difosfato Quinasas NM23/genética , Nucleósido Difosfato Quinasas NM23/metabolismo , Metástasis de la Neoplasia , ProteómicaRESUMEN
The mechanisms of how cancer cells are selected and evolve to establish distant metastatic colonies remain unclear. Tumor heterogeneity and lack of biomarkers are some of the most difficult challenges in cancer biology and treatment. Here using mouse models for triple-negative breast cancer (TNBC) metastasis, we report heterogeneous expression of DNA methyltransferase 3B (DNMT3B) in both mouse and human primary tumors. High levels of DNMT3B were correlated with poor clinical outcomes in multiple human breast cancer datasets. Mechanistically, clonal cells with high DNMT3B (DNMT3BH) showed higher vimentin (VIM) expression and displayed enhanced epithelial-to-mesenchymal transition capacity. Deletion of VIM diminished the metastatic phenotype of DNMT3BH cells. Importantly, in preclinical mouse models in which the primary tumors were surgically removed, perioperative targeting of DNMT3B in combination with chemotherapy markedly suppressed tumor recurrence and metastasis. Our studies identify DNMT3B-mediated transcription regulation as an important mediator of tumor heterogeneity and show that DNMT3B is critical for tumor invasion and metastasis, reinforcing its potential as a target for treating metastatic disease. IMPLICATIONS: Our findings of transcriptome changes mediated by DNMT3B provide new mechanistic insight for intratumor heterogeneity and chemoresistance, and therapeutic targeting of DNMT3B in combination with chemotherapy offer additional treatment options for metastatic disease especially for patients with TNBC.
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ADN (Citosina-5-)-Metiltransferasas , Neoplasias de la Mama Triple Negativas , Animales , Humanos , Ratones , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Recurrencia Local de Neoplasia/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , ADN Metiltransferasa 3BRESUMEN
The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells.
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Apoptosis , Neoplasias de la Mama , Canales de Cloruro , Glutarredoxinas , Peróxido de Hidrógeno , Mitocondrias , Proteínas Mitocondriales , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Femenino , Eliminación de Gen , Glutarredoxinas/metabolismo , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Necrosis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Superóxidos/metabolismoRESUMEN
Background: Secretoglobin (SCGB) 3A2 is a novel bioactive molecule with anti-inflammatory and anti-fibrotic activities. SCGB3A2 also promotes the maturation of bronchial divergence and the lungs during embryonic development. However, much remains unknown concerning the roles of SCGB3A2 in diseases associated with aging. Methods: The lungs of Scgb3a2-knockout (KO) mice and their wild-type (WT) littermates were subjected to histological analysis, Victoria blue staining to evaluate of elastic fibers, and lung morphometric analysis during the postnatal period (birth to 8 weeks) and during aging (8 weeks to 2 years). Their spleens were also histologically evaluated. The expression of lung surfactant protein (SP) mRNAs was examined by quantitative reverse transcriptase-polymerase chain reaction. RNA sequencing (RNAseq) analysis was performed on 3-month-old KO and WT mouse lungs. Results: The alveolar spaces of KO mice continuously expanded between 0.5 and 2 years of age, accompanied by increases of the mean linear intercept and destructive index. KO mouse lungs displayed inflammation associated with lymphocyte aggregate starting at 1 year of age, and the inflammation was worse than that of WT mouse lungs. A high number of lymphoma-like cells were presented in 2-year-old KO mouse lungs. White pulp fusion was detected in the spleens of both WT and KO mice older than 0.5 years; however, the fusion was more severe in KO mice than in WT mice. The expression of surfactant protein (SP)-A, SP-B, SP-C, and SP-D mRNAs in KO mouse lungs decreased with age, and after 1 year of age, the expression of most SPs was significantly lower in KO mice than in WT mice. RNAseq demonstrated that the expression of immune system-related genes was highly altered in KO mouse lungs. Conclusion: SCGB3A2 may be required for maintaining homeostasis and immune activity in the lungs during aging. SCGB3A2 deficiency might increase the risk of emphysema of the lung.
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Enfisema , Linfoma , Enfermedad Pulmonar Obstructiva Crónica , Envejecimiento , Animales , Femenino , Humanos , Inflamación/metabolismo , Pulmón/metabolismo , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Noqueados , Embarazo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secretoglobinas/genética , Secretoglobinas/metabolismo , Tensoactivos/metabolismoRESUMEN
We integrated ESCC expression and GWAS genotyping, to investigate eQTL and somatic DNA segment alterations, including somatic copy number alteration, allelic imbalance (AI), and loss of heterozygosity (LOH) in ESCC. First, in eQTL analysis, we used a classical approach based on genotype data from GWAS and expression signals in normal tissue samples, and then used a modified approach based on fold change in the tumor vs. normal samples. We focused on the genes in three pathways: inflammation, DNA repair, and immunity. Among the significant (p < 0.05) SNP-probe pairs from classical and modified eQTL analyses, 24 genes were shared by the two approaches, including 18 genes that showed the same numbers of SNPs and probes and 6 genes that had the different numbers of SNPs and probes. For these 18 genes, we found 28 SNP−probe pairs were correlated in opposite directions in the two approaches, indicating an intriguing difference between the classical and modified eQTL approaches. Second, we analyzed the somatic DNA segment alterations. Across the 24 genes, abnormal gene expression on mRNA arrays was seen in 19−95% of cases and 26−78% showed somatic DNA segment alterations on Affymetrix GeneChip Human Mapping Arrays. The results suggested that this strategy could identify gene expression and somatic DNA segment alterations for biological markers (genes) by combining classical and modified eQTLs and somatic DNA evaluation on SNP arrays. Thus, this study approach may allow us to understand functionality indicative of potentially relevant biomarkers in ESCC.
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We introduce HUNTRESS, a computational method for mutational intratumor heterogeneity inference from noisy genotype matrices derived from single-cell sequencing data, the running time of which is linear with the number of cells and quadratic with the number of mutations. We prove that, under reasonable conditions, HUNTRESS computes the true progression history of a tumor with high probability. On simulated and real tumor sequencing data, HUNTRESS is demonstrated to be faster than available alternatives with comparable or better accuracy. Additionally, the progression histories of tumors inferred by HUNTRESS on real single-cell sequencing datasets agree with the best known evolution scenarios for the associated tumors.
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Neoplasias , Humanos , Neoplasias/genética , Análisis de Secuencia , MutaciónRESUMEN
Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.
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Neoplasias de la Mama/patología , Heterocromatina/metabolismo , Mecanotransducción Celular/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Factor de Unión a CCCTC/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Citoesqueleto/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Adhesiones Focales/genética , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , Metiltransferasas/metabolismo , Ratones , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas Nucleares/metabolismo , Polimorfismo Genético , Pronóstico , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Microambiente TumoralRESUMEN
The tumor microenvironment (TME) influences cancer progression and therapy response. Therefore, understanding what regulates the TME immune compartment is vital. Here we show that microbiota signals program mononuclear phagocytes in the TME toward immunostimulatory monocytes and dendritic cells (DCs). Single-cell RNA sequencing revealed that absence of microbiota skews the TME toward pro-tumorigenic macrophages. Mechanistically, we show that microbiota-derived stimulator of interferon genes (STING) agonists induce type I interferon (IFN-I) production by intratumoral monocytes to regulate macrophage polarization and natural killer (NK) cell-DC crosstalk. Microbiota modulation with a high-fiber diet triggered the intratumoral IFN-I-NK cell-DC axis and improved the efficacy of immune checkpoint blockade (ICB). We validated our findings in individuals with melanoma treated with ICB and showed that the predicted intratumoral IFN-I and immune compositional differences between responder and non-responder individuals can be transferred by fecal microbiota transplantation. Our study uncovers a mechanistic link between the microbiota and the innate TME that can be harnessed to improve cancer therapies.
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Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Microbiota , Monocitos/metabolismo , Microambiente Tumoral , Akkermansia/efectos de los fármacos , Akkermansia/fisiología , Animales , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Fibras de la Dieta/farmacología , Fosfatos de Dinucleósidos/administración & dosificación , Fosfatos de Dinucleósidos/farmacología , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunomodulación/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Melanoma/inmunología , Melanoma/patología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microbiota/efectos de los fármacos , Monocitos/efectos de los fármacos , Fagocitos/efectos de los fármacos , Fagocitos/metabolismo , Transcripción Genética/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacosRESUMEN
Cellular senescence is a well-documented response to oncogene activation in many tissues. Multiple pathways are invoked to achieve senescence indicating its importance to counteract the transforming activities of oncogenic stimulation. We now report that the Rho-associated protein kinase (ROCK) signaling pathway is a critical regulator of oncogene-induced senescence in skin carcinogenesis. Transformation of mouse keratinocytes with oncogenic RAS upregulates ROCK activity and initiates a senescence response characterized by cell enlargement, growth inhibition, upregulation of senescence associated ß-galactosidase (SAßgal) expression, and release of multiple pro-inflammatory factors comprising the senescence-associated secretory phenotype (SASP). The addition of the ROCK inhibitor Y-27632 and others prevents these senescence responses and maintains proliferating confluent RAS transformed keratinocyte cultures indefinitely. Mechanistically, oncogenic RAS transformation is associated with upregulation of cell cycle inhibitors p15Ink4b , p16Ink4a , and p19Arf and downregulation of p-AKT, all of which are reversed by Y-27632. RNA-seq analysis of Y-27632 treated RAS-transformed keratinocytes indicated that the inhibitor reduced growth-inhibitory gene expression profiles and maintained expression of proliferative pathways. Y-27632 also reduced the expression of NF-κB effector genes and the expression of IκBζ downstream mediators. The senescence inhibition from Y-27632 was reversible, and upon its removal, senescence reoccurred in vitro with rapid upregulation of cell cycle inhibitors, SASP expression, and cell detachment. Y-27632 treated cultured RAS-keratinocytes formed tumors in the absence of the inhibitor when placed in skin orthografts suggesting that factors in the tumor microenvironment can overcome the drive to senescence imparted by overactive ROCK activity.
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Amidas/administración & dosificación , Transformación Celular Neoplásica/efectos de los fármacos , Queratinocitos/citología , Piridinas/administración & dosificación , Neoplasias Cutáneas/patología , Proteínas ras/genética , Quinasas Asociadas a rho/metabolismo , Amidas/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/trasplante , Ratones , Piridinas/farmacología , Análisis de Secuencia de ARN , Transducción de Señal , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismoRESUMEN
PURPOSE: Breast cancer diagnosed in young patients is often aggressive. Because primary breast tumors from young and older patients have similar mutational patterns, we hypothesized that the young host microenvironment promotes more aggressive metastatic disease. EXPERIMENTAL DESIGN: Triple-negative or luminal B breast cancer cell lines were injected into young and older mice side-by-side to quantify lung, liver, and brain metastases. Young and older mouse brains, metastatic and naïve, were analyzed by flow cytometry. Immune populations were depleted using antibodies or a colony-stimulating factor-1 receptor (CSF-1R) inhibitor, and brain metastasis assays were conducted. Effects on myeloid populations, astrogliosis, and the neuroinflammatory response were determined. RESULTS: Brain metastases were 2- to 4-fold higher in young as compared with older mouse hosts in four models of triple-negative or luminal B breast cancer; no age effect was observed on liver or lung metastases. Aged brains, naïve or metastatic, contained fewer resident CNS myeloid cells. Use of a CSF-1R inhibitor to deplete myeloid cells, including both microglia and infiltrating macrophages, preferentially reduced brain metastasis burden in young mice. Downstream effects of CSF-1R inhibition in young mice resembled that of an aged brain in terms of myeloid numbers, induction of astrogliosis, and Semaphorin 3A secretion within the neuroinflammatory response. CONCLUSIONS: Host microenvironmental factors contribute to the aggressiveness of triple-negative and luminal B breast cancer brain metastasis. CSF-1R inhibitors may hold promise for young brain metastasis patients.
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Neoplasias Encefálicas/secundario , Células Mieloides , Neoplasias de la Mama Triple Negativas/patología , Factores de Edad , Animales , Línea Celular Tumoral , Sistema Nervioso Central/citología , Humanos , Ratones , Receptor de Factor Estimulante de Colonias de Macrófagos/fisiologíaRESUMEN
Metabolic regulation is critical for the maintenance of pluripotency and the survival of embryonic stem cells (ESCs). The transcription factor Tfcp2l1 has emerged as a key factor for the naïve pluripotency of ESCs. Here, we report an unexpected role of Tfcp2l1 in metabolic regulation in ESCs-promoting the survival of ESCs through regulating fatty acid oxidation (FAO) under metabolic stress. Tfcp2l1 directly activates many metabolic genes in ESCs. Deletion of Tfcp2l1 leads to an FAO defect associated with upregulation of glucose uptake, the TCA cycle, and glutamine catabolism. Mechanistically, Tfcp2l1 activates FAO by inducing Cpt1a, a rate-limiting enzyme transporting free fatty acids into the mitochondria. ESCs with defective FAO are sensitive to cell death induced by glycolysis inhibition and glutamine deprivation. Moreover, the Tfcp2l1-Cpt1a-FAO axis promotes the survival of quiescent ESCs and diapause-like blastocysts induced by mTOR inhibition. Thus, our results reveal how ESCs orchestrate pluripotent and metabolic programs to ensure their survival in response to metabolic stress.
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Células Madre Embrionarias , Metabolismo de los Lípidos , Ácidos Grasos , Oxidación-Reducción , Estrés FisiológicoRESUMEN
PURPOSE: Molecular similarities have been reported between basal-like breast cancer (BLBC) and high-grade serous ovarian cancer (HGSOC). To date, there have been no prognostic biomarkers that can provide risk stratification and inform treatment decisions for both BLBC and HGSOC. In this study, we developed a molecular signature for risk stratification in BLBC and further validated this signature in HGSOC. METHODS: RNA-seq data was downloaded from The Cancer Genome Atlas (TCGA) project for 190 BLBC and 314 HGSOC patients. Analyses of differentially expressed genes between recurrent vs. non-recurrent cases were performed using different bioinformatics methods. Gene Signature was established using weighted linear combination of gene expression levels. Their prognostic performance was evaluated using survival analysis based on progression-free interval (PFI) and disease-free interval (DFI). RESULTS: 63 genes were differentially expressed between 18 recurrent and 40 non-recurrent BLBC patients by two different methods. The recurrence index (RI) calculated from this 63-gene signature significantly stratified BLBC patients into two risk groups with 38 and 152 patients in the low-risk (RI-Low) and high-risk (RI-High) groups, respectively (p = 0.0004 and 0.0023 for PFI and DFI, respectively). Similar performance was obtained in the HGSOC cohort (p = 0.0131 and 0.004 for PFI and DFI, respectively). Multivariate Cox regression adjusting for age, grade, and stage showed that the 63-gene signature remained statistically significant in stratifying HGSOC patients (p = 0.0005). CONCLUSION: A gene signature was identified to predict recurrence in BLBC and HGSOC patients. With further validation, this signature may provide an additional prognostic tool for clinicians to better manage BLBC, many of which are triple-negative and HGSOC patients who are currently difficult to treat.
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Neoplasias de la Mama , Cistadenocarcinoma Seroso , Neoplasias Ováricas , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Cistadenocarcinoma Seroso/genética , Femenino , Humanos , Recurrencia Local de Neoplasia/genética , Neoplasias Ováricas/genética , PronósticoRESUMEN
MOTIVATION: Recent advances in single-cell sequencing (SCS) offer an unprecedented insight into tumor emergence and evolution. Principled approaches to tumor phylogeny reconstruction via SCS data are typically based on general computational methods for solving an integer linear program, or a constraint satisfaction program, which, although guaranteeing convergence to the most likely solution, are very slow. Others based on Monte Carlo Markov Chain or alternative heuristics not only offer no such guarantee, but also are not faster in practice. As a result, novel methods that can scale up to handle the size and noise characteristics of emerging SCS data are highly desirable to fully utilize this technology. RESULTS: We introduce PhISCS-BnB (phylogeny inference using SCS via branch and bound), a branch and bound algorithm to compute the most likely perfect phylogeny on an input genotype matrix extracted from an SCS dataset. PhISCS-BnB not only offers an optimality guarantee, but is also 10-100 times faster than the best available methods on simulated tumor SCS data. We also applied PhISCS-BnB on a recently published large melanoma dataset derived from the sublineages of a cell line involving 20 clones with 2367 mutations, which returned the optimal tumor phylogeny in <4 h. The resulting phylogeny agrees with and extends the published results by providing a more detailed picture on the clonal evolution of the tumor. AVAILABILITY AND IMPLEMENTATION: https://github.com/algo-cancer/PhISCS-BnB. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.