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Hypothyroidism is commonly detected in patients with medulloblastoma (MB). However, whether thyroid hormone (TH) contributes to MB pathogenicity remains undetermined. Here, we find that TH plays a critical role in promoting tumor cell differentiation. Reduction in TH levels frees the TH receptor, TRα1, to bind to EZH2 and repress expression of NeuroD1, a transcription factor that drives tumor cell differentiation. Increased TH reverses EZH2-mediated repression of NeuroD1 by abrogating the binding of EZH2 and TRα1, thereby stimulating tumor cell differentiation and reducing MB growth. Importantly, TH-induced differentiation of tumor cells is not restricted by the molecular subgroup of MB, suggesting that TH can be used to broadly treat MB subgroups. These findings establish an unprecedented association between TH signaling and MB pathogenicity, providing solid evidence for TH as a promising modality for MB treatment.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Proteína Potenciadora del Homólogo Zeste 2 , Meduloblastoma , Hormonas Tiroideas , Meduloblastoma/patología , Meduloblastoma/metabolismo , Meduloblastoma/genética , Humanos , Diferenciación Celular/efectos de los fármacos , Animales , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Ratones , Hormonas Tiroideas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Neoplasias Cerebelosas/patología , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/tratamiento farmacológico , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptores alfa de Hormona Tiroidea/metabolismo , Receptores alfa de Hormona Tiroidea/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Understanding pancreatic cancer biology is fundamental for identifying new targets and for developing more effective therapies. In particular, the contribution of the stromal microenvironment to pancreatic cancer tumorigenesis requires further exploration. Here, we report the stromal roles of the synaptic protein Netrin G1 Ligand (NGL-1) in pancreatic cancer, uncovering its pro-tumor functions in cancer-associated fibroblasts and in immune cells. We observed that the stromal expression of NGL-1 inversely correlated with patients' overall survival. Moreover, germline knockout (KO) mice for NGL-1 presented decreased tumor burden, with a microenvironment that is less supportive of tumor growth. Of note, tumors from NGL-1 KO mice produced less immunosuppressive cytokines and displayed an increased percentage of CD8 + T cells than those from control mice, while preserving the physical structure of the tumor microenvironment. These effects were shown to be mediated by NGL-1 in both immune cells and in the local stroma, in a TGF-ß-dependent manner. While myeloid cells lacking NGL-1 decreased the production of immunosuppressive cytokines, NGL-1 KO T cells showed increased proliferation rates and overall polyfunctionality compared to control T cells. CAFs lacking NGL-1 were less immunosuppressive than controls, with overall decreased production of pro-tumor cytokines and compromised ability to inhibit CD8 + T cells activation. Mechanistically, these CAFs downregulated components of the TGF-ß pathway, AP-1 and NFAT transcription factor families, resulting in a less tumor-supportive phenotype. Finally, targeting NGL-1 genetically or using a functionally antagonistic small peptide phenocopied the effects of chemotherapy, while modulating the immunosuppressive tumor microenvironment (TME), rather than eliminating it. We propose NGL-1 as a new local stroma and immunomodulatory molecule, with pro-tumor roles in pancreatic cancer. Statement of Significance: Here we uncovered the pro-tumor roles of the synaptic protein NGL-1 in the tumor microenvironment of pancreatic cancer, defining a new target that simultaneously modulates tumor cell, fibroblast, and immune cell functions. This study reports a new pathway where NGL-1 controls TGF-ß, AP-1 transcription factor members and NFAT1, modulating the immunosuppressive microenvironment in pancreatic cancer. Our findings highlight NGL-1 as a new stromal immunomodulator in pancreatic cancer.
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Prostate cancer (PCa) is the most common cancer diagnosed in men worldwide and the second leading cause of cancer-related deaths in US males in 2022. Prostate cancer also represents the second highest cancer mortality disparity between non-Hispanic blacks and whites. However, there is a relatively small number of prostate normal and cancer cell lines compared to other cancers. To identify the molecular basis of PCa progression, it is important to have prostate epithelial cell (PrEC) lines as karyotypically normal as possible. Our lab recently developed a novel methodology for the rapid and efficient immortalization of normal human PrEC that combines simultaneous CRISPR-directed inactivation of CDKN2A exon 2 (which directs expression of p16INK4A and p14ARF) and ectopic expression of an hTERT transgene. To optimize this methodology to generate immortalized lines with minimal genetic alterations, we sought to target exon 1α of the CDKN2A locus so that p16INK4A expression is ablated while p14ARF expression remains unaltered. Here we describe the establishment of two cell lines: one with the above-mentioned p16INK4A only loss, and a second line targeting both products in the CDKN2A locus. We characterize the potential lineage origin of these new cell lines along with our previously obtained clones, revealing distinct gene expression signatures. Based on the analyses of protein markers and RNA expression signatures, these cell lines are most closely related to a subpopulation of basal prostatic cells. Given the simplicity of this one-step methodology and the fact that it uses only the minimal genetic alterations necessary for immortalization, it should also be suitable for the establishment of cell lines from primary prostate tumor samples, an urgent need given the limited number of available prostate cancer cell lines.
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BACKGROUND & AIMS: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. METHODS: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining. RESULTS: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. CONCLUSIONS: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.
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Autofagia , Proliferación Celular , Homeostasis , Ratones Noqueados , Organoides , Animales , Ratones , Organoides/metabolismo , Esófago/patología , Esófago/citología , Esófago/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteína 7 Relacionada con la Autofagia/genética , 4-Nitroquinolina-1-Óxido , Autorrenovación de las Células , Mucosa Esofágica/patología , Mucosa Esofágica/metabolismo , Mucosa Esofágica/citología , Análisis de la Célula IndividualRESUMEN
Hypothyroidism is commonly detected in patients with medulloblastoma (MB). A possible link between thyroid hormone (TH) signaling and MB pathogenicity has not been reported. Here, we find that TH plays a critical role in promoting tumor cell differentiation. Reduction in TH levels frees the TH receptor, TRα1, to bind to EZH2 and repress expression of NeuroD1, a transcription factor that drives tumor cell differentiation. Increased TH reverses EZH2-mediated repression of NeuroD1 by abrogating the binding of EZH2 and TRα1, thereby stimulating tumor cell differentiation and reducing MB growth. Importantly, TH-induced differentiation of tumor cells is not restricted by the molecular subgroup of MB. These findings establish an unprecedented association between TH signaling and MB pathogenicity, providing solid evidence for TH as a promising modality for MB treatment.
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Activating innate immunity in cancer cells through cytoplasmic nucleic acid sensing pathways, a phenomenon known as "viral mimicry," has emerged as an effective strategy to convert immunologically "cold" tumors into "hot." Through a curated CRISPR-based screen of RNA helicases, we identified DExD/H-box helicase 9 (DHX9) as a potent repressor of double-stranded RNA (dsRNA) in small cell lung cancers (SCLC). Depletion of DHX9 induced accumulation of cytoplasmic dsRNA and triggered tumor-intrinsic innate immunity. Intriguingly, ablating DHX9 also induced aberrant accumulation of R-loops, which resulted in an increase of DNA damage-derived cytoplasmic DNA and replication stress in SCLCs. In vivo, DHX9 deletion promoted a decrease in tumor growth while inducing a more immunogenic tumor microenvironment, invigorating responsiveness to immune-checkpoint blockade. These findings suggest that DHX9 is a crucial repressor of tumor-intrinsic innate immunity and replication stress, representing a promising target for SCLC and other "cold" tumors in which genomic instability contributes to pathology. SIGNIFICANCE: One promising strategy to trigger an immune response within tumors and enhance immunotherapy efficacy is by inducing endogenous "virus-mimetic" nucleic acid accumulation. Here, we identify DHX9 as a viral-mimicry-inducing factor involved in the suppression of double-stranded RNAs and R-loops and propose DHX9 as a novel target to enhance antitumor immunity. See related commentary by Chiappinelli, p. 389. This article is featured in Selected Articles from This Issue, p. 384.
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Neoplasias Pulmonares , Ácidos Nucleicos , Carcinoma Pulmonar de Células Pequeñas , Humanos , Carcinoma Pulmonar de Células Pequeñas/genética , Interferones , Neoplasias Pulmonares/genética , Inmunidad Innata , ARN Bicatenario , Microambiente Tumoral , Proteínas de Neoplasias , ARN Helicasas DEAD-box/genéticaRESUMEN
Background & Aims: Autophagy has been demonstrated to play roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelium under homeostatic conditions. Methods: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histological and biochemical analyses. We FACS sorted esophageal basal cells based upon fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID, then subjected these cells to transmission electron microscopy, image flow cytometry, 3D organoid assays, RNA-Sequencing (RNA-Seq), and cell cycle analysis. 3D organoids were subjected to passaging, single cell (sc) RNA-Seq, cell cycle analysis, and immunostaining. Results: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells. Esophageal basal cells with high AV level (Cyto-ID High ) displayed limited organoid formation capability upon initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-ID Low ). RNA-Seq suggested increased autophagy in Cyto- ID High esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. scRNA-Seq of 3D organoids generated by Cyto-ID Low and Cyto- ID High cells identified expansion of 3 cell populations, enrichment of G2/M-associated genes, and aberrant localization of cell cycle-associated genes beyond basal cell populations in the Cyto- ID High group. Ki67 expression was also increased in organoids generated by Cyto-ID High cells, including in cells beyond the basal cell layer. Squamous epithelial-specific autophagy inhibition induced significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Conclusions: High AV level identifies esophageal epithelium with limited proliferation and enhanced self-renewal capacity that contributes to maintenance of the esophageal proliferation- differentiation gradient in vivo .
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Induced pluripotent stem cells (iPSCs) have enormous potential in producing human tissues endlessly. We previously reported that type V collagen (COL5), a pancreatic extracellular matrix protein, promotes islet development and maturation from iPSCs. In this study, we identified a bioactive peptide domain of COL5, WWASKS, through bioinformatic analysis of decellularized pancreatic ECM (dpECM)-derived collagens. RNA-sequencing suggests that WWASKS induces the formation of pancreatic endocrine progenitors while suppressing the development of other types of organs. The expressions of hypoxic genes were significantly downregulated in the endocrine progenitors formed under peptide stimulation. Furthermore, we unveiled an enhancement of iPSC-derived islets' (i-islets) glucose sensitivity under peptide stimulation. These i-islets secrete insulin in a glucose responsive manner. They were comprised of α, ß, δ, and γ cells and were assembled into a tissue architecture similar to that of human islets. Mechanistically, the peptide is able to activate the canonical Wnt signaling pathway, permitting the translocation of ß-catenin from the cytoplasm to the nucleus for pancreatic progenitor development. Collectively, for the first time, we demonstrated that an ECM-derived peptide dictates iPSC fate toward the generation of endocrine progenitors and subsequent islet organoids.
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Introduction: Under homeostatic conditions, esophageal epithelium displays a proliferation/differentiation gradient that is generated as proliferative basal cells give rise to suprabasal cells then terminally differentiated superficial cells. This proliferation/differentiation gradient is often perturbed in esophageal pathologies. Basal cell hyperplasia may occur in patients with gastroesophageal reflux disease (GERD), a condition in which acid from the stomach enters the esophagus, or eosinophilic esophagitis (EoE), an emerging form of food allergy. While GERD is a primary risk factor for esophageal cancer, epidemiological data suggests that EoE patients do not develop esophageal cancer. Methods: In order to investigate the impact of EoE and esophageal cancer specifically on the cellular landscape of esophageal epithelium, we perform single cell RNA-sequencing in murine models of EoE and esophageal cancer, specifically esophageal squamous cell carcinoma (ESCC). We further evaluate modules of co-expressed genes in EoE- and ESCC-enriched epithelial cell clusters. Finally, we pair EoE and ESCC murine models to examine the functional relationship between these pathologies. Results: In mice with either EoE or ESCC, we find expansion of cell populations as compared to normal esophageal epithelium. In mice with EoE, we detect distinct expansion of 4 suprabasal populations coupled with depletion of 2 basal populations. By contrast, mice with ESCC display unique expansion of 2 basal populations and 1 suprabasal population, as well as depletion of 2 suprabasal populations. Senescence, glucocorticoid receptor signaling, and granulocyte-macrophage colony-stimulating factor pathways are associated with EoE-enriched clusters while pathways associated with cell proliferation and metabolism are identified in ESCC-enriched clusters. Finally, our in vivo data demonstrate that exposure to EoE inflammation limits tumor burden of esophageal carcinogenesis. Discussion: Our findings provide the first functional investigation of the relationship between EoE and esophageal cancer and suggest that esophageal epithelial remodeling events occurring in response to EoE inflammation may limit esophageal carcinogenesis. This investigation may have future implications for leveraging allergic inflammation-associated alterations in epithelial biology to prevent and/or treat esophageal cancer.
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The significance of alveolar epithelial type 2 (AT2) cell proliferation for lung alveolar epithelial homeostasis and regeneration after injury has been widely accepted. However, the heterogeneity of AT2 cell population for cell proliferation capacity remains disputed. By single-cell RNA sequencing and genetic lineage labeling using the Ki67 knock-in mouse model, we map all proliferative AT2 cells in homeostatic and regenerating murine lungs after injury induced by Streptococcus pneumoniae infection. The proliferative AT2 cell population displays a unique transcriptional program, which is regulated by activating transcription factor 3 (ATF3) and thyroid hormone receptor alpha (THRA) transcription factors. Overexpression of these two transcription factors in AT2 cells promoted AT2 cell proliferation and improved lung function after injury. These results indicate that increased expression of ATF3 and THRA at the onset of lung epithelial regeneration is required to permit rapid AT2 cell proliferation and hence progression through the recovery of lung epithelium.
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Although morphologic progression coupled with expression of specific molecular markers has been characterized along the esophageal squamous differentiation gradient, the molecular heterogeneity within cell types along this trajectory has yet to be classified at the single cell level. To address this knowledge gap, we perform single cell RNA-sequencing of 44,679 murine esophageal epithelial, to identify 11 distinct cell populations as well as pathways alterations along the basal-superficial axis and in each individual population. We evaluate the impact of aging upon esophageal epithelial cell populations and demonstrate age-associated mitochondrial dysfunction. We compare single cell transcriptomic profiles in 3D murine organoids and human esophageal biopsies with that of murine esophageal epithelium. Finally, we employ pseudotemporal trajectory analysis to develop a working model of cell fate determination in murine esophageal epithelium. These studies provide comprehensive molecular perspective on the cellular heterogeneity of murine esophageal epithelium in the context of homeostasis and aging.
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Neoplasias Esofágicas , Transcriptoma , Animales , Células Epiteliales , Epitelio/metabolismo , Neoplasias Esofágicas/patología , Esófago/patología , Humanos , Ratones , Análisis de la Célula Individual , Transcriptoma/genéticaRESUMEN
Epstein-Barr virus (EBV) persists in human B-cells by maintaining its chromatinized episomes within the nucleus. We have previously shown that cellular factor Poly [ADP-ribose] polymerase 1 (PARP1) binds the EBV genome, stabilizes CTCF binding at specific loci, and that PARP1 enzymatic activity correlates with maintaining a transcriptionally active latency program. To better understand PARP1's role in regulating EBV latency, here we functionally characterize the effect of PARP enzymatic inhibition on episomal structure through in situ HiC mapping, generating a complete 3D structure of the EBV genome. We also map intragenomic contact changes after PARP inhibition to global binding of chromatin looping factors CTCF and cohesin across the EBV genome. We find that PARP inhibition leads to fewer total unique intragenomic interactions within the EBV episome, yet new chromatin loops distinct from the untreated episome are also formed. This study also illustrates that PARP inhibition alters gene expression at the regions where chromatin looping is most effected. We observe that PARP1 inhibition does not alter cohesin binding sites but does increase its frequency of binding at those sites. Taken together, these findings demonstrate that PARP has an essential role in regulating global EBV chromatin structure and latent gene expression.
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Proteínas de Ciclo Celular/genética , Cromatina/química , Proteínas Cromosómicas no Histona/genética , Mapeo Cromosómico/métodos , Genoma Viral , Herpesvirus Humano 4/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Linfocitos B/patología , Linfocitos B/virología , Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Regulación de la Expresión Génica , Herpesvirus Humano 4/efectos de los fármacos , Herpesvirus Humano 4/crecimiento & desarrollo , Herpesvirus Humano 4/inmunología , Interacciones Huésped-Patógeno , Humanos , Modelos Moleculares , Ftalazinas/farmacología , Piperazinas/farmacología , Plásmidos/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Unión Proteica , Transducción de Señal , Transcripción Genética , Latencia del Virus/genética , CohesinasRESUMEN
Because loss of the NF2 tumor suppressor gene results in p21-activated kinase (Pak) activation, PAK inhibitors hold promise for the treatment of NF2-deficient tumors. To test this possibility, we asked if loss of Pak2, a highly expressed group I PAK member, affects the development of malignant mesothelioma in Nf2;Cdkn2a-deficient (NC) mice and the growth properties of NC mesothelioma cells in culture. In vivo, deletion of Pak2 resulted in a markedly decreased incidence and delayed onset of both pleural and peritoneal malignant mesotheliomas in NC mice. In vitro, Pak2 deletion decreased malignant mesothelioma cell viability, migration, clonogenicity, and spheroid formation. RNA-sequencing analysis demonstrated downregulated expression of Hedgehog and Wnt pathway genes in NC;Pak2-/- mesothelioma cells versus NC;Pak2+/+ mesothelioma cells. Targeting of the Hedgehog signaling component Gli1 or its target gene Myc inhibited cell viability and spheroid formation in NC;P+/+ mesothelioma cells. Kinome profiling uncovered kinase changes indicative of EMT in NC;Pak2-/- mesothelioma cells, suggesting that Pak2-deficient malignant mesotheliomas can adapt by reprogramming their kinome in the absence of Pak activity. The identification of such compensatory pathways offers opportunities for rational combination therapies to circumvent resistance to anti-PAK drugs. IMPLICATIONS: We provide evidence supporting a role for PAK inhibitors in treating NF2-deficient tumors. NF2-deficient tumors lacking Pak2 eventually adapt by kinome reprogramming, presenting opportunities for combination therapies to bypass anti-PAK drug resistance.
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Mesotelioma Maligno , Mesotelioma , Animales , Proteínas Hedgehog/genética , Humanos , Mesotelioma/tratamiento farmacológico , Mesotelioma/genética , Ratones , Vía de Señalización Wnt , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
Acute damage to the heart, as in the case of myocardial infarction (MI), triggers a robust inflammatory response to the sterile injury that is part of a complex and highly organized wound-healing process. Cortical bone stem cell (CBSC) therapy after MI has been shown to reduce adverse structural and functional remodeling of the heart after MI in both mouse and swine models. The basis for these CBSC treatment effects on wound healing are unknown. The present experiments show that CBSCs secrete paracrine factors known to have immunomodulatory properties, most notably macrophage colony-stimulating factor (M-CSF) and transforming growth factor-ß, but not IL-4. CBSC therapy increased the number of galectin-3+ macrophages, CD4+ T cells, and fibroblasts in the heart while decreasing apoptosis in an in vivo swine model of MI. Macrophages treated with CBSC medium in vitro polarized to a proreparative phenotype are characterized by increased CD206 expression, increased efferocytic ability, increased IL-10, TGF-ß, and IL-1RA secretion, and increased mitochondrial respiration. Next generation sequencing revealed a transcriptome significantly different from M2a or M2c macrophage phenotypes. Paracrine factors from CBSC-treated macrophages increased proliferation, decreased α-smooth muscle actin expression, and decreased contraction by fibroblasts in vitro. These data support the idea that CBSCs are modulating the immune response to MI to favor cardiac repair through a unique macrophage polarization that ultimately reduces cell death and alters fibroblast populations that may result in smaller scar size and preserved cardiac geometry and function.NEW & NOTEWORTHY Cortical bone stem cell (CBSC) therapy after myocardial infarction alters the inflammatory response to cardiac injury. We found that cortical bone stem cell therapy induces a unique macrophage phenotype in vitro and can modulate macrophage/fibroblast cross talk.
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Mediadores de Inflamación/metabolismo , Activación de Macrófagos , Macrófagos/metabolismo , Infarto del Miocardio/cirugía , Miocardio/metabolismo , Comunicación Paracrina , Trasplante de Células Madre , Células Madre/metabolismo , Cicatrización de Heridas , Animales , Apoptosis , Células Cultivadas , Hueso Cortical/citología , Modelos Animales de Enfermedad , Femenino , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibrosis , Humanos , Macrófagos/inmunología , Ratones Endogámicos C57BL , Infarto del Miocardio/genética , Infarto del Miocardio/inmunología , Infarto del Miocardio/metabolismo , Miocardio/inmunología , Fenotipo , Transducción de Señal , Porcinos , Porcinos Enanos , Linfocitos T/inmunología , Linfocitos T/metabolismo , TranscriptomaRESUMEN
Homeobox genes control body patterning and cell-fate decisions during development. The homeobox genes consist of many families, only some of which have been investigated regarding a possible role in tumorigenesis. Dysregulation of HOX family genes have been widely implicated in cancer etiology. DLX homeobox genes, which belong to the NK-like family, exert dual roles in development and cancer. The DLX genes are the key transcription factors involved in regulating the development of craniofacial structures in vertebrates. The three DLX bigenes have overlapping expression in the branchial arches. Disruption of DLX function has destructive consequences in organogenesis and is associated with certain congenital disorders in humans. The role of DLX genes in oncogenesis is only beginning to emerge. DLX2 diminishes cellular senescence by regulating p53 function, whereas DLX4 has been associated with metastasis in breast cancer. In human ovarian cancer cells, DLX5 is essential for regulating AKT signaling, thereby promoting cell proliferation and survival. We previously implicated Dlx5 as an oncogene in murine T-cell lymphoma driven by a constitutively active form of Akt2. In this mouse model, overexpression of Dlx5 was caused by a chromosomal rearrangement that juxtaposed the Tcr-beta promoter region near the Dlx5 locus. Moreover, transgenic mice overexpressing Dlx5, specifically in immature T-cells, develop spontaneous thymic lymphomas. Oncogenesis in this mouse model involves binding of Dlx5 to the Notch1 and Notch3 gene loci to activate their transcription. Dlx5 also cooperates with Akt signaling to accelerate lymphomagenesis by activating Wnt signaling. We also discuss the fact that human DLX5 is aberrantly expressed in several human malignancies.
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The transcription factor ThPOK (encoded by the Zbtb7b gene) controls homeostasis and differentiation of mature helper T cells, while opposing their differentiation to CD4+ intraepithelial lymphocytes (IELs) in the intestinal mucosa. Thus CD4 IEL differentiation requires ThPOK transcriptional repression via reactivation of the ThPOK transcriptional silencer element (SilThPOK). In the present study, we describe a new autoregulatory loop whereby ThPOK binds to the SilThPOK to maintain its own long-term expression in CD4 T cells. Disruption of this loop in vivo prevents persistent ThPOK expression, leads to genome-wide changes in chromatin accessibility and derepresses the colonic regulatory T (Treg) cell gene expression signature. This promotes selective differentiation of naive CD4 T cells into GITRloPD-1loCD25lo (Triplelo) Treg cells and conversion to CD4+ IELs in the gut, thereby providing dominant protection from colitis. Hence, the ThPOK autoregulatory loop represents a key mechanism to physiologically control ThPOK expression and T cell differentiation in the gut, with potential therapeutic relevance.
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Proteínas de Unión al ADN/metabolismo , Linfocitos Intraepiteliales/citología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Reguladores/citología , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/inmunología , Colitis/inmunología , Colitis/prevención & control , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Femenino , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Factores de Transcripción/genética , Transcripción Genética/genéticaRESUMEN
Metabolic reprogramming is a common feature of many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators that promote AML metabolic reprogramming and the benefits conferred to leukemia cells by these metabolic changes remain largely unknown. We report that the transcription factor ATF3 coordinates serine and nucleotide metabolism to maintain cell cycling, survival, and the differentiation blockade in AML. Analysis of mouse and human AML models demonstrate that ATF3 directly activates the transcription of genes encoding key enzymatic regulators of serine synthesis, one-carbon metabolism, and de novo purine and pyrimidine synthesis. Total steady-state polar metabolite and heavy isotope tracing analyses show that ATF3 inhibition reduces de novo serine synthesis, impedes the incorporation of serine-derived carbons into newly synthesized purines, and disrupts pyrimidine metabolism. Importantly, exogenous nucleotide supplementation mitigates the anti-leukemia effects of ATF3 inhibition. Together, these findings reveal the dependence of AML on ATF3-regulated serine and nucleotide metabolism.
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Factor de Transcripción Activador 3/metabolismo , Ciclo Celular , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleótidos/metabolismo , Serina/metabolismo , Factor de Transcripción Activador 3/genética , Línea Celular Tumoral , Humanos , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Nucleótidos/genética , Serina/genéticaRESUMEN
Ferroptosis is associated with lipid hydroperoxides generated by the oxidation of polyunsaturated acyl chains. Lipid hydroperoxides are reduced by glutathione peroxidase 4 (GPX4) and GPX4 inhibitors induce ferroptosis. However, the therapeutic potential of triggering ferroptosis in cancer cells with polyunsaturated fatty acids is unknown. Here, we identify conjugated linoleates including α-eleostearic acid (αESA) as ferroptosis inducers. αESA does not alter GPX4 activity but is incorporated into cellular lipids and promotes lipid peroxidation and cell death in diverse cancer cell types. αESA-triggered death is mediated by acyl-CoA synthetase long-chain isoform 1, which promotes αESA incorporation into neutral lipids including triacylglycerols. Interfering with triacylglycerol biosynthesis suppresses ferroptosis triggered by αESA but not by GPX4 inhibition. Oral administration of tung oil, naturally rich in αESA, to mice limits tumor growth and metastasis with transcriptional changes consistent with ferroptosis. Overall, these findings illuminate a potential approach to ferroptosis, complementary to GPX4 inhibition.
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Coenzima A Ligasas/metabolismo , Ferroptosis , Ácidos Linolénicos/metabolismo , Neoplasias de la Mama Triple Negativas/enzimología , Neoplasias de la Mama Triple Negativas/fisiopatología , Animales , Muerte Celular , Coenzima A Ligasas/genética , Humanos , Ratones , Ratones Endogámicos NOD , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell-mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SIGNIFICANCE: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.See related commentary by Sherman, p. 230.This article is highlighted in the In This Issue feature, p. 211.
Asunto(s)
Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Netrinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Terapia de Inmunosupresión , Apoyo Nutricional , Microambiente TumoralRESUMEN
BACKGROUND: Immortalization of primary prostate epithelial cells (PrEC) with just hTERT expression is particularly inefficient in the absence of DNA tumor viral proteins or p16INK4A knockdown. MATERIALS AND METHODS: Here, we describe the establishment of immortalized normal prostate epithelial cell line models using CRISPR technology to inactivate the CDKN2A locus concomitantly with ectopic expression of an hTERT transgene. RESULTS: Using this approach, we have obtained immortal cell clones that exhibit fundamental characteristics of normal cells, including diploid genomes, near normal karyotypes, normal p53 and pRB cell responses, the ability to form non-invasive spheroids, and a non-transformed phenotype. Based on marker expression, these clones are of basal cell origin. CONCLUSIONS: Use of this approach resulted in the immortalization of independent clones of PrEC that retained normal characteristics, were stable, and non-transformed. Thus, this approach could be used for the immortalization of normal primary prostate cells. This technique could also be useful for establishing cell lines from prostate tumor tissues of different tumor grades and/or from patients of diverse ethnicities to generate cell line models that facilitate the study of the molecular basis of disease disparity.