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The replication of eukaryotic chromosomes is organized temporally and spatially within the nucleus through epigenetic regulation of replication origin function. The characteristic initiation timing of specific origins is thought to reflect their chromatin environment or sub-nuclear positioning, however the mechanism remains obscure. Here we show that the yeast Forkhead transcription factors, Fkh1 and Fkh2, are global determinants of replication origin timing. Forkhead regulation of origin timing is independent of local levels or changes of transcription. Instead, we show that Fkh1 and Fkh2 are required for the clustering of early origins and their association with the key initiation factor Cdc45 in G1 phase, suggesting that Fkh1 and Fkh2 selectively recruit origins to emergent replication factories. Fkh1 and Fkh2 bind Fkh-activated origins, and interact physically with ORC, providing a plausible mechanism to cluster origins. These findings add a new dimension to our understanding of the epigenetic basis for differential origin regulation and its connection to chromosomal domain organization.
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Proteínas de Ciclo Celular/metabolismo , Factores de Transcripción Forkhead/metabolismo , Origen de Réplica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al ADN/metabolismo , Fase G1 , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
We have combined a machine-learning approach with other strategies to optimize knockout efficiency with the CRISPR/Cas9 system. In addition, we have developed a multiplexed sgRNA expression strategy that promotes the functional ablation of single genes and allows for combinatorial targeting. These strategies have been combined to design and construct a genome-wide, sequence-verified, arrayed CRISPR library. This resource allows single-target or combinatorial genetic screens to be carried out at scale in a multiplexed or arrayed format. By conducting parallel loss-of-function screens, we compare our approach to existing sgRNA design and expression strategies.
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Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Endonucleasas/genética , Silenciador del Gen , Marcación de Gen/métodos , ARN Guía de Kinetoplastida/genética , Algoritmos , Proteínas Asociadas a CRISPR/metabolismo , Endonucleasas/metabolismo , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células K562 , Aprendizaje Automático , ARN Guía de Kinetoplastida/metabolismo , TransfecciónRESUMEN
This corrects the article DOI: 10.1038/nature25465.
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Using a functional model of breast cancer heterogeneity, we previously showed that clonal sub-populations proficient at generating circulating tumour cells were not all equally capable of forming metastases at secondary sites. A combination of differential expression and focused in vitro and in vivo RNA interference screens revealed candidate drivers of metastasis that discriminated metastatic clones. Among these, asparagine synthetase expression in a patient's primary tumour was most strongly correlated with later metastatic relapse. Here we show that asparagine bioavailability strongly influences metastatic potential. Limiting asparagine by knockdown of asparagine synthetase, treatment with l-asparaginase, or dietary asparagine restriction reduces metastasis without affecting growth of the primary tumour, whereas increased dietary asparagine or enforced asparagine synthetase expression promotes metastatic progression. Altering asparagine availability in vitro strongly influences invasive potential, which is correlated with an effect on proteins that promote the epithelial-to-mesenchymal transition. This provides at least one potential mechanism for how the bioavailability of a single amino acid could regulate metastatic progression.
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Asparagina/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Metástasis de la Neoplasia/patología , Animales , Asparaginasa/metabolismo , Asparaginasa/uso terapéutico , Asparagina/deficiencia , Aspartatoamoníaco Ligasa/genética , Aspartatoamoníaco Ligasa/metabolismo , Disponibilidad Biológica , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Masculino , Ratones , Invasividad Neoplásica/patología , Pronóstico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Interferencia de ARN , Reproducibilidad de los ResultadosRESUMEN
Bladder cancer (BC) has a 70% telomerase reverse transcriptase (TERT or hTERT in humans) promoter mutation prevalence, commonly at -124 base pairs, and this is associated with increased hTERT expression and poor patient prognosis. We inserted a green fluorescent protein (GFP) tag in the mutant hTERT promoter allele to create BC cells expressing an hTERT-GFP fusion protein. These cells were used in a fluorescence-activated cell sorting-based pooled CRISPR-Cas9 Kinome knockout genetic screen to identify tripartite motif containing 28 (TRIM28) and TRIM24 as regulators of hTERT expression. TRIM28 activates, while TRIM24 suppresses, hTERT transcription from the mutated promoter allele. TRIM28 is recruited to the mutant promoter where it interacts with TRIM24, which inhibits its activity. Phosphorylation of TRIM28 through the mTOR complex 1 (mTORC1) releases it from TRIM24 and induces hTERT transcription. TRIM28 expression promotes in vitro and in vivo BC cell growth and stratifies BC patient outcome. mTORC1 inhibition with rapamycin analog Ridaforolimus suppresses TRIM28 phosphorylation, hTERT expression, and cell viability. This study may lead to hTERT-directed cancer therapies with reduced effects on normal progenitor cells.
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Mutación/genética , Regiones Promotoras Genéticas/genética , Telomerasa/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Proteína 28 que Contiene Motivos Tripartito/genética , Neoplasias de la Vejiga Urinaria/genética , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Regulación Enzimológica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Células Madre/patologíaRESUMEN
PIWI proteins and their associated piRNAs protect germ cells from the activity of mobile genetic elements. Two classes of piRNAsprimary and secondaryare defined by their mechanisms of biogenesis. Primary piRNAs are processed directly from transcripts of piRNA cluster loci, whereas secondary piRNAs are generated in an adaptive amplification loop, termed the ping-pong cycle. In mammals, piRNA populations are dynamic, shifting as male germ cells develop. Embryonic piRNAs consist of both primary and secondary species and are mainly directed toward transposons. In meiotic cells, the piRNA population is transposon-poor and largely restricted to primary piRNAs derived from pachytene piRNA clusters. The transition from the embryonic to the adult piRNA pathway is not well understood. Here we show that RNF17 shapes adult meiotic piRNA content by suppressing the production of secondary piRNAs. In the absence of RNF17, ping-pong occurs inappropriately in meiotic cells. Ping-pong initiates piRNA responses against not only transposons but also protein-coding genes and long noncoding RNAs, including genes essential for germ cell development. Thus, the sterility of Rnf17 mutants may be a manifestation of a small RNA-based autoimmune reaction.
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Proteínas Argonautas/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Testículo/fisiopatología , Factores de Transcripción/metabolismo , Animales , Proteínas Argonautas/genética , Elementos Transponibles de ADN/genética , Técnicas de Inactivación de Genes , Masculino , Meiosis/genética , Ratones , Mutación , ARN Interferente Pequeño/metabolismo , Testículo/metabolismo , Factores de Transcripción/genéticaRESUMEN
Accurate staging of bladder cancer assists in identifying optimal treatment (e.g., transurethral resection vs. radical cystectomy vs. bladder preservation). However, currently, about one-third of patients are over-staged and one-third are under-staged. There is a pressing need for a more accurate staging modality to evaluate patients with bladder cancer to assist clinical decision-making. We hypothesize that MRI/RNA-seq-based radiogenomics and artificial intelligence can more accurately stage bladder cancer. A total of 40 magnetic resonance imaging (MRI) and matched formalin-fixed paraffin-embedded (FFPE) tissues were available for analysis. Twenty-eight (28) MRI and their matched FFPE tissues were available for training analysis, and 12 matched MRI and FFPE tissues were used for validation. FFPE samples were subjected to bulk RNA-seq, followed by bioinformatics analysis. In the radiomics, several hundred image-based features from bladder tumors in MRI were extracted and analyzed. Overall, the model obtained mean sensitivity, specificity, and accuracy of 94%, 88%, and 92%, respectively, in differentiating intra- vs. extra-bladder cancer. The proposed model demonstrated improvement in the three matrices by 17%, 33%, and 25% and 17%, 16%, and 17% as compared to the genetic- and radiomic-based models alone, respectively. The radiogenomics of bladder cancer provides insight into discriminative features capable of more accurately staging bladder cancer. Additional studies are underway.
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Inteligencia Artificial , Neoplasias de la Vejiga Urinaria , Humanos , RNA-Seq , Neoplasias de la Vejiga Urinaria/diagnóstico por imagen , Neoplasias de la Vejiga Urinaria/genética , Imagen por Resonancia Magnética , MúsculosRESUMEN
The strength of conclusions drawn from RNAi-based studies is heavily influenced by the quality of tools used to elicit knockdown. Prior studies have developed algorithms to design siRNAs. However, to date, no established method has emerged to identify effective shRNAs, which have lower intracellular abundance than transfected siRNAs and undergo additional processing steps. We recently developed a multiplexed assay for identifying potent shRNAs and used this method to generate â¼250,000 shRNA efficacy data points. Using these data, we developed shERWOOD, an algorithm capable of predicting, for any shRNA, the likelihood that it will elicit potent target knockdown. Combined with additional shRNA design strategies, shERWOOD allows the ab initio identification of potent shRNAs that specifically target the majority of each gene's multiple transcripts. We validated the performance of our shRNA designs using several orthogonal strategies and constructed genome-wide collections of shRNAs for humans and mice based on our approach.
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ARN Interferente Pequeño/genética , Programas Informáticos , Algoritmos , Secuencia de Bases , Línea Celular Tumoral , Simulación por Computador , Secuencia de Consenso , Técnicas de Silenciamiento del Gen , Humanos , MicroARNs/genética , Modelos Genéticos , Datos de Secuencia MolecularRESUMEN
Cancer metastasis requires that primary tumour cells evolve the capacity to intravasate into the lymphatic system or vasculature, and extravasate into and colonize secondary sites. Others have demonstrated that individual cells within complex populations show heterogeneity in their capacity to form secondary lesions. Here we develop a polyclonal mouse model of breast tumour heterogeneity, and show that distinct clones within a mixed population display specialization, for example, dominating the primary tumour, contributing to metastatic populations, or showing tropism for entering the lymphatic or vasculature systems. We correlate these stable properties to distinct gene expression profiles. Those clones that efficiently enter the vasculature express two secreted proteins, Serpine2 and Slpi, which were necessary and sufficient to program these cells for vascular mimicry. Our data indicate that these proteins not only drive the formation of extravascular networks but also ensure their perfusion by acting as anticoagulants. We propose that vascular mimicry drives the ability of some breast tumour cells to contribute to distant metastases while simultaneously satisfying a critical need of the primary tumour to be fed by the vasculature. Enforced expression of SERPINE2 and SLPI in human breast cancer cell lines also programmed them for vascular mimicry, and SERPINE2 and SLPI were overexpressed preferentially in human patients that had lung-metastatic relapse. Thus, these two secreted proteins, and the phenotype they promote, may be broadly relevant as drivers of metastatic progression in human cancer.
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Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/patología , Endotelio Vascular/patología , Metástasis de la Neoplasia/patología , Animales , Anticoagulantes/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Células Clonales/metabolismo , Células Clonales/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Femenino , Perfilación de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Metástasis de la Neoplasia/genética , Recurrencia , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Análisis de Secuencia de ADN , Serpina E2/metabolismoRESUMEN
PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism to provide essential protection for germ-cell genomes against the activity of mobile genetic elements. piRNA populations comprise a molecular definition of transposons, which permits them to distinguish transposons from host genes and selectively silence them. piRNAs can be generated in two distinct ways, forming either primary or secondary piRNAs. Primary piRNAs come from discrete genomic loci, termed piRNA clusters, and seem to be derived from long, single-stranded precursors. The biogenesis of primary piRNAs involves at least two nucleolytic steps. An unknown enzyme cleaves piRNA cluster transcripts to generate monophosphorylated piRNA 5' ends. piRNA 3' ends are probably formed by exonucleolytic trimming, after a piRNA precursor is loaded into its PIWI partner. Secondary piRNAs arise during the adaptive 'ping-pong' cycle, with their 5' termini being formed by the activity of PIWIs themselves. A number of proteins have been implicated genetically in primary piRNA biogenesis. One of these, Drosophila melanogaster Zucchini, is a member of the phospholipase-D family of phosphodiesterases, which includes both phospholipases and nucleases. Here we produced a dimeric, soluble fragment of the mouse Zucchini homologue (mZuc; also known as PLD6) and show that it possesses single-strand-specific nuclease activity. A crystal structure of mZuc at 1.75 Å resolution indicates greater architectural similarity to phospholipase-D family nucleases than to phospholipases. Together, our data suggest that the Zucchini proteins act in primary piRNA biogenesis as nucleases, perhaps generating the 5' ends of primary piRNAs.
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Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Fosfolipasa D/química , Fosfolipasa D/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Ratones , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , ARN Interferente Pequeño/biosíntesis , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Electricidad Estática , Especificidad por SustratoRESUMEN
In higher eukaryotes, heritable gene silencing is associated with histone deacetylation and late replication timing. In Saccharomyces cerevisiae, the histone deacetylase Rpd3 regulates gene expression and also modulates replication timing; however, these mechanisms have been suggested to be independent, and no global association has been found between replication timing and gene expression levels. Using 5-Bromo-2'-deoxyuridine (BrdU) incorporation to generate genome-wide replication profiles, we identified >100 late-firing replication origins that are regulated by Rpd3L, which is specifically targeted to promoters to silence transcription. Rpd3S, which recompacts chromatin after transcription, plays a primary role at only a handful of origins, but subtly influences initiation timing globally. The ability of these functionally distinct Rpd3 complexes to affect replication initiation timing supports the idea that histone deacetylation directly influences initiation timing. Accordingly, loss of Rpd3 function results in higher levels of histone H3 and H4 acetylation surrounding Rpd3-regulated origins, and these origins show a significant association with Rpd3 chromatin binding and gene regulation, supporting a general link between histone acetylation, replication timing, and control of gene expression in budding yeast. Our results also reveal a novel and complementary genomic map of Rpd3L- and Rpd3S-regulated chromosomal loci.
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Replicación del ADN/genética , Genoma Fúngico/genética , Histona Desacetilasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Hidroxiurea/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Regiones Promotoras Genéticas , Origen de Réplica/efectos de los fármacos , Proteínas Represoras/metabolismo , Factores de TiempoRESUMEN
Spared regions of the damaged central nervous system undergo dynamic remodeling and exhibit a remarkable potential for therapeutic exploitation. Here, lesion-remote astrocytes (LRAs), which interact with viable neurons, glia and neural circuitry, undergo reactive transformations whose molecular and functional properties are poorly understood. Using multiple transcriptional profiling methods, we interrogated LRAs from spared regions of mouse spinal cord following traumatic spinal cord injury (SCI). We show that LRAs acquire a spectrum of molecularly distinct, neuroanatomically restricted reactivity states that evolve after SCI. We identify transcriptionally unique reactive LRAs in degenerating white matter that direct the specification and function of local microglia that clear lipid-rich myelin debris to promote tissue repair. Fueling this LRA functional adaptation is Ccn1 , which encodes for a secreted matricellular protein. Loss of astrocyte CCN1 leads to excessive, aberrant activation of local microglia with (i) abnormal molecular specification, (ii) dysfunctional myelin debris processing, and (iii) impaired lipid metabolism, culminating in blunted debris clearance and attenuated neurological recovery from SCI. Ccn1 -expressing white matter astrocytes are specifically induced by local myelin damage and generated in diverse demyelinating disorders in mouse and human, pointing to their fundamental, evolutionarily conserved role in white matter repair. Our findings show that LRAs assume regionally divergent reactivity states with functional adaptations that are induced by local context-specific triggers and influence disorder outcome. Astrocytes tile the central nervous system (CNS) where they serve vital roles that uphold healthy nervous system function, including regulation of synapse development, buffering of neurotransmitters and ions, and provision of metabolic substrates 1 . In response to diverse CNS insults, astrocytes exhibit disorder-context specific transformations that are collectively referred to as reactivity 2-5 . The characteristics of regionally and molecularly distinct reactivity states are incompletely understood. The mechanisms through which distinct reactivity states arise, how they evolve or resolve over time, and their consequences for local cell function and CNS disorder progression remain enigmatic. Immediately adjacent to CNS lesions, border-forming astrocytes (BFAs) undergo transcriptional reprogramming and proliferation to form a neuroprotective barrier that restricts inflammation and supports axon regeneration 6-9 . Beyond the lesion, spared but dynamic regions of the injured CNS exhibit varying degrees of synaptic circuit remodeling and progressive cellular responses to secondary damage that have profound consequences for neural repair and recovery 10,11 . Throughout these cytoarchitecturally intact, but injury-reactive regions, lesion-remote astrocytes (LRAs) intermingle with neurons and glia, undergo little to no proliferation, and exhibit varying degrees of cellular hypertrophy 7,12,13 . The molecular and functional properties of LRAs remain grossly undefined. Therapeutically harnessing spared regions of the injured CNS will require a clearer understanding of the accompanying cellular and molecular landscape. Here, we leveraged integrative transcriptional profiling methodologies to identify multiple spatiotemporally resolved, molecularly distinct states of LRA reactivity within the injured spinal cord. Computational modeling of LRA-mediated heterotypic cell interactions, astrocyte-specific conditional gene deletion, and multiple mouse models of acute and chronic CNS white matter degeneration were used to interrogate a newly identified white matter degeneration-reactive astrocyte subtype. We define how this reactivity state is induced and its role in governing the molecular and functional specification of local microglia that clear myelin debris from the degenerating white matter to promote repair.
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Strategies are needed to better identify patients that will benefit from immunotherapy alone or who may require additional therapies like chemotherapy or radiotherapy to overcome resistance. Here we employ single-cell transcriptomics and spatial proteomics to profile triple negative breast cancer biopsies taken at baseline, after one cycle of pembrolizumab, and after a second cycle of pembrolizumab given with radiotherapy. Non-responders lack immune infiltrate before and after therapy and exhibit minimal therapy-induced immune changes. Responding tumors form two groups that are distinguishable by a classifier prior to therapy, with one showing high major histocompatibility complex expression, evidence of tertiary lymphoid structures, and displaying anti-tumor immunity before treatment. The other responder group resembles non-responders at baseline and mounts a maximal immune response, characterized by cytotoxic T cell and antigen presenting myeloid cell interactions, only after combination therapy, which is mirrored in a murine model of triple negative breast cancer.
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Neoplasias de la Mama Triple Negativas , Humanos , Animales , Ratones , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/radioterapia , Anticuerpos Monoclonales Humanizados/uso terapéutico , Terapia Combinada , InmunoterapiaRESUMEN
Bladder cancer (BLCA) is more common in men but more aggressive in women. Sex-based differences in cancer biology are commonly studied using a murine model with BLCA generated by N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). While tumors in the BBN model have been profiled, these profiles provide limited information on the tumor microenvironment. Here, we applied single-cell RNA sequencing to characterize cell-type specific transcriptional differences between male and female BBN-induced tumors. We found proportional and gene expression differences in epithelial and non-epithelial subpopulations between male and female tumors. Expression of several genes predicted sex-specific survival in several human BLCA datasets. We identified novel and clinically relevant sex-specific transcriptional signatures including immune cells in the tumor microenvironment and it validated the relevance of the BBN model for studying sex differences in human BLCA. This work highlights the importance of considering sex as a biological variable in the development of new and accurate cancer markers.
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Estrogen and progesterone have been extensively studied in the mammary gland, but the molecular effects of androgen remain largely unexplored. Transgender men are recorded as female at birth but identify as male and may undergo gender-affirming androgen therapy to align their physical characteristics and gender identity. Here we perform single-cell-resolution transcriptome, chromatin, and spatial profiling of breast tissues from transgender men following androgen therapy. We find canonical androgen receptor gene targets are upregulated in cells expressing the androgen receptor and that paracrine signaling likely drives sex-relevant androgenic effects in other cell types. We also observe involution of the epithelium and a spatial reconfiguration of immune, fibroblast, and vascular cells, and identify a gene regulatory network associated with androgen-induced fat loss. This work elucidates the molecular consequences of androgen activity in the human breast at single-cell resolution.
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Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference.
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Neoplasias , Humanos , Terapia Combinada , Neoplasias/radioterapia , Neoplasias/tratamiento farmacológico , InmunoterapiaRESUMEN
Neoadjuvant chemotherapy (NAC) prior to surgery and immune checkpoint therapy (ICT) have revolutionized bladder cancer management. However, stratification of patients that would benefit most from these modalities remains a major clinical challenge. Here, we combine single nuclei RNA sequencing with spatial transcriptomics and single-cell resolution spatial proteomic analysis of human bladder cancer to identify an epithelial subpopulation with therapeutic response prediction ability. These cells express Cadherin 12 (CDH12, N-Cadherin 2), catenins, and other epithelial markers. CDH12-enriched tumors define patients with poor outcome following surgery with or without NAC. In contrast, CDH12-enriched tumors exhibit superior response to ICT. In all settings, patient stratification by tumor CDH12 enrichment offers better prediction of outcome than currently established bladder cancer subtypes. Molecularly, the CDH12 population resembles an undifferentiated state with inherently aggressive biology including chemoresistance, likely mediated through progenitor-like gene expression and fibroblast activation. CDH12-enriched cells express PD-L1 and PD-L2 and co-localize with exhausted T-cells, possibly mediated through CD49a (ITGA1), providing one explanation for ICT efficacy in these tumors. Altogether, this study describes a cancer cell population with an intriguing diametric response to major bladder cancer therapeutics. Importantly, it also provides a compelling framework for designing biomarker-guided clinical trials.
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Cadherinas/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Inmunoterapia/métodos , Neoplasias de la Vejiga Urinaria/terapia , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proteínas Relacionadas con las Cadherinas , Cadherinas/metabolismo , Cateninas/genética , Cateninas/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Estimación de Kaplan-Meier , Terapia Neoadyuvante/métodos , Evaluación de Resultado en la Atención de Salud , Proteómica/métodos , RNA-Seq/métodos , Linfocitos T/metabolismo , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismo , Vejiga Urinaria/cirugía , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/cirugíaRESUMEN
Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients.
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Factor de Transcripción Activador 4/metabolismo , Asparagina/metabolismo , Mitocondrias/metabolismo , Animales , Asparagina/farmacología , Ácido Aspártico/deficiencia , Ácido Aspártico/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dieta/veterinaria , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Metformina/farmacología , Metformina/uso terapéutico , Ratones , Ratones Endogámicos NOD , Mitocondrias/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/mortalidad , Neoplasias/patología , Nucleótidos/metabolismo , Tasa de SupervivenciaRESUMEN
BACKGROUND: Partially methylated domains (PMDs) are a hallmark of epigenomes in reproducible and specific biological contexts, including cancer cells, the placenta, and cultured cell lines. Existing methods for deciding whether PMDs exist in a sample, as well as their identification, are few, often tailored to specific biological questions, and require high coverage samples for accurate identification. RESULTS: In this study, we outline a set of axioms that take a step towards a functional definition for PMDs, describe an improved method for comparable PMD detection across samples with substantially differing sequencing depths, and refine the decision criteria for whether a sample contains PMDs using a data-driven approach. Applying our method to 267 methylomes from 7 species, we corroborated recent results regarding the general association between replication timing and PMD state, and report identification of several reproducibly "escapee" genes within late-replicating domains that escape the reduced expression and hypomethylation of their immediate genomic neighborhood. We also explored the discordant PMD state of orthologous genes between human and mouse, and observed a directional association of PMD state with gene expression and local gene density. CONCLUSIONS: Our improved method makes low sequencing depth, population-level studies of PMD variation possible and our results further refine the model of PMD formation as one where sequence context and regional epigenomic features both play a role in gradual genome-wide hypomethylation.