RESUMEN
Metastases arise from subsets of cancer cells that disseminate from the primary tumour1,2. The ability of cancer cells to thrive in a new tissue site is influenced by genetic and epigenetic changes that are important for disease initiation and progression, but these factors alone do not predict if and where cancers metastasize3,4. Specific cancer types metastasize to consistent subsets of tissues, suggesting that primary tumour-associated factors influence where cancers can grow. We find primary and metastatic pancreatic tumours have metabolic similarities and that the tumour-initiating capacity and proliferation of both primary-derived and metastasis-derived cells is favoured in the primary site relative to the metastatic site. Moreover, propagating cells as tumours in the lung or the liver does not enhance their relative ability to form large tumours in those sites, change their preference to grow in the primary site, nor stably alter aspects of their metabolism relative to primary tumours. Primary liver and lung cancer cells also exhibit a preference to grow in their primary site relative to metastatic sites. These data suggest cancer tissue of origin influences both primary and metastatic tumour metabolism and may impact where cancer cells can metastasize.
RESUMEN
Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions.
Asunto(s)
Diferenciación Celular , Replicación del ADN , Replicación del ADN/genética , Animales , Humanos , Diferenciación Celular/genética , Ratones , Nucleótidos/metabolismo , Nucleótidos/genética , Linaje de la Célula/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/metabolismo , Fase S/genética , Transducción de SeñalRESUMEN
Blocking the import of nutrients essential for cancer cell proliferation represents a therapeutic opportunity, but it is unclear which transporters to target. Here we report a CRISPR interference/activation screening platform to systematically interrogate the contribution of nutrient transporters to support cancer cell proliferation in environments ranging from standard culture media to tumours. We applied this platform to identify the transporters of amino acids in leukaemia cells and found that amino acid transport involves high bidirectional flux dependent on the microenvironment composition. While investigating the role of transporters in cystine starved cells, we uncovered a role for serotonin uptake in preventing ferroptosis. Finally, we identified transporters essential for cell proliferation in subcutaneous tumours and found that levels of glucose and amino acids can restrain proliferation in that environment. This study establishes a framework for systematically identifying critical cellular nutrient transporters, characterizing their function and exploring how the tumour microenvironment impacts cancer metabolism.
Asunto(s)
Proliferación Celular , Microambiente Tumoral , Humanos , Animales , Sistemas CRISPR-Cas , Nutrientes/metabolismo , Línea Celular Tumoral , Transporte Biológico , Glucosa/metabolismo , Aminoácidos/metabolismo , Serotonina/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Ratones , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente EspaciadasRESUMEN
Amino acids are required for cell growth and proliferation, but it remains unclear when and how amino acid availability impinges on the proliferation-quiescence decision. Here, we used time-lapse microscopy and single-cell tracking of cyclin-dependent kinase 2 (CDK2) activity to assess the response of individual cells to withdrawal of single amino acids and found strikingly different cell-cycle effects depending on the amino acid. For example, upon leucine withdrawal, MCF10A cells complete two cell cycles and then enter a CDK2-low quiescence, whereas lysine withdrawal causes immediate cell-cycle stalling. Methionine withdrawal triggers a restriction point phenotype similar to serum starvation or Mek inhibition: upon methionine withdrawal, cells complete their current cell cycle and enter a CDK2-low quiescence after mitosis. Modulation of restriction point regulators p21/p27 or cyclin D1 enables short-term rescue of proliferation under methionine and leucine withdrawal, and to a lesser extent lysine withdrawal, revealing a checkpoint connecting nutrient signaling to cell-cycle entry.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Aminoácidos , Leucina , Lisina , Ciclo Celular , Quinasa 2 Dependiente de la Ciclina/metabolismo , Puntos de Control del Ciclo Celular , Mitosis , Metionina , Quinasas CDC2-CDC28/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismoRESUMEN
Nucleotide metabolism supports RNA synthesis and DNA replication to enable cell growth and division. Nucleotide depletion can inhibit cell growth and proliferation, but how cells sense and respond to changes in the relative levels of individual nucleotides is unclear. Moreover, the nucleotide requirement for biomass production changes over the course of the cell cycle, and how cells coordinate differential nucleotide demands with cell cycle progression is not well understood. Here we find that excess levels of individual nucleotides can inhibit proliferation by disrupting the relative levels of nucleotide bases needed for DNA replication and impeding DNA replication. The resulting purine and pyrimidine imbalances are not sensed by canonical growth regulatory pathways like mTORC1, Akt and AMPK signalling cascades, causing excessive cell growth despite inhibited proliferation. Instead, cells rely on replication stress signalling to survive during, and recover from, nucleotide imbalance during S phase. We find that ATR-dependent replication stress signalling is activated during unperturbed S phases and promotes nucleotide availability to support DNA replication. Together, these data reveal that imbalanced nucleotide levels are not detected until S phase, rendering cells reliant on replication stress signalling to cope with this metabolic problem and disrupting the coordination of cell growth and division.
Asunto(s)
Replicación del ADN , Nucleótidos , Ciclo Celular/genética , División Celular , Replicación del ADN/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Nucleótidos/genética , Nucleótidos/metabolismo , Fase SRESUMEN
Folate metabolism can be an effective target for cancer treatment. However, standard cell culture conditions utilize folic acid, a non-physiological folate source for most tissues. We find that the enzyme that couples folate and methionine metabolic cycles, methionine synthase, is required for cancer cell proliferation and tumour growth when 5-methyl tetrahydrofolate (THF), the major folate found in circulation, is the extracellular folate source. In such physiological conditions, methionine synthase incorporates 5-methyl THF into the folate cycle to maintain intracellular levels of the folates needed for nucleotide production. 5-methyl THF can sustain intracellular folate metabolism in the absence of folic acid. Therefore, cells exposed to 5-methyl THF are more resistant to methotrexate, an antifolate drug that specifically blocks folic acid incorporation into the folate cycle. Together, these data argue that the environmental folate source has a profound effect on folate metabolism, determining how both folate cycle enzymes and antifolate drugs impact proliferation.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Neoplasias/metabolismo , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos/genética , Ácido Fólico/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Metotrexato/farmacología , Neoplasias/etiología , Neoplasias/patología , Tetrahidrofolatos/metabolismoRESUMEN
Tumors are composed of many different cell types including cancer cells, fibroblasts, and immune cells. Dissecting functional metabolic differences between cell types within a mixed population can be challenging due to the rapid turnover of metabolites relative to the time needed to isolate cells. To overcome this challenge, we traced isotope-labeled nutrients into macromolecules that turn over more slowly than metabolites. This approach was used to assess differences between cancer cell and fibroblast metabolism in murine pancreatic cancer organoid-fibroblast co-cultures and tumors. Pancreatic cancer cells exhibited increased pyruvate carboxylation relative to fibroblasts, and this flux depended on both pyruvate carboxylase and malic enzyme 1 activity. Consequently, expression of both enzymes in cancer cells was necessary for organoid and tumor growth, demonstrating that dissecting the metabolism of specific cell populations within heterogeneous systems can identify dependencies that may not be evident from studying isolated cells in culture or bulk tissue.
Tumors contain a mixture of many different types of cells, including cancer cells and non-cancer cells. The interactions between these two groups of cells affect how the cancer cells use nutrients, which, in turn, affects how fast these cells grow and divide. Furthermore, different cell types may use nutrients in diverse ways to make other molecules known as metabolites that the cell needs to survive. Fibroblasts are a subset of non-cancer cells that are typically found in tumors and can help them form. Separating fibroblasts from cancer cells in a tumor takes a lot longer than the chemical reactions in each cell of the tumor that produce and use up nutrients, also known as the cell's metabolism. Therefore, measuring the levels of glucose (the sugar that is the main energy source for cells) and other metabolites in each tumor cell after separating them does not necessarily provide accurate information about the tumor cell's metabolism. This makes it difficult to study how cancer cells and fibroblasts use nutrients differently. Lau et al. have developed a strategy to study the metabolism of cancer cells and fibroblasts in tumors. Mice with tumors in their pancreas were provided glucose that had been labelled using biochemical techniques. As expected, when the cell processed the glucose, the label was transferred into metabolites that got used up very quickly. But the label also became incorporated into larger, more stable molecules, such as proteins. Unlike the small metabolites, these larger molecules do not change in the time it takes to separate the cancer cells from the fibroblasts. Lau et al. sorted cells from whole pancreatic tumors and analyzed large, stable molecules that can incorporate the label from glucose in cancer cells and fibroblasts. The experiments showed that, in cancer cells, these molecules were more likely to have labeling patterns that are characteristic of two specific enzymes called pyruvate carboxylase and malic enzyme 1. This suggests that these enzymes are more active in cancer cells. Lau et al. also found that pancreatic cancer cells needed these two enzymes to metabolize glucose and to grow into large tumors. Pancreatic cancer is one of the most lethal cancers and current therapies offer limited benefit to many patients. Therefore, it is important to develop new drugs to treat this disease. Understanding how cancer cells and non-cancer cells in pancreatic tumors use nutrients differently is important for developing drugs that only target cancer cells.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Tumors exhibit altered metabolism compared to normal tissues. Many cancers upregulate expression of serine synthesis pathway enzymes, and some tumors exhibit copy-number gain of the gene encoding the first enzyme in the pathway, phosphoglycerate dehydrogenase (PHGDH). However, whether increased serine synthesis promotes tumor growth and how serine synthesis benefits tumors is controversial. Here, we demonstrate that increased PHGDH expression promotes tumor progression in mouse models of melanoma and breast cancer, human tumor types that exhibit PHGDH copy-number gain. We measure circulating serine levels and find that PHGDH expression is necessary to support cell proliferation at lower physiological serine concentrations. Increased dietary serine or high PHGDH expression is sufficient to increase intracellular serine levels and support faster tumor growth. Together, these data suggest that physiological serine availability restrains tumor growth and argue that tumors arising in serine-limited environments acquire a fitness advantage by upregulating serine synthesis pathway enzymes.
Asunto(s)
Proliferación Celular , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Serina/biosíntesis , Animales , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Neoplasias/genética , Serina/metabolismoRESUMEN
Limitation for amino acids is thought to regulate translation in mammalian cells primarily by signaling through the kinases mTORC1 and GCN2. We find that a selective loss of arginine tRNA charging during limitation for arginine regulates translation through ribosome pausing at two of six arginine codons. Surprisingly, limitation for leucine, an essential and abundant amino acid in protein, results in little or no ribosome pausing. Chemical and genetic perturbation of mTORC1 and GCN2 signaling revealed that their robust response to leucine limitation prevents ribosome pausing, while an insufficient response to arginine limitation leads to loss of tRNA charging and ribosome pausing. Ribosome pausing decreases protein production and triggers premature ribosome termination without reducing mRNA levels. Together, our results suggest that amino acids that are not optimally sensed by the mTORC1 and GCN2 pathways still regulate translation through an evolutionarily conserved mechanism based on codon-specific ribosome pausing.
Asunto(s)
Factor 2 Eucariótico de Iniciación/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/fisiología , Biosíntesis de Proteínas/fisiología , Aminoácidos/metabolismo , Animales , Arginina/metabolismo , Codón/metabolismo , Leucina/metabolismo , Mamíferos/genética , Extensión de la Cadena Peptídica de Translación/genética , Extensión de la Cadena Peptídica de Translación/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , ARN/metabolismo , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Natural products remain an important source of new therapeutics for emerging drug-resistant pathogens like Candida albicans, which particularly affects immunocompromised patients. A bioactive 3-decalinoyltetramic acid, pyrrolocin A, was isolated from extracts of a novel Amazonian fungal endophyte, E6927E, of the Diaporthales family. The structure of the natural product was solved using NMR and CD spectroscopy and it is structurally related to the fungal setins, equisetin and phomasetin, which are well-characterized tetramic acid antibiotics specific for Gram-positive organisms. We show that the compound inhibits growth of Staphylococcus aureus and Enterococcus faecalis. It shows selective and potent bioactivity against fungal strains, with an MIC of 4 µg/mL for C. albicans, 100 µg/mL for Aspergillus sp. and greater than 100 µg/mL for Saccharomyces cerevisiae. Further, the compound is less toxic to mammalian cells (IC50 = 150 µg/mL), with an inhibitory concentration greater than forty times that for C. albicans. Pyrrolocin A retained potent activity against eight out of seventeen strains of clinical Candida sp. isolates tested.
Asunto(s)
Ascomicetos/química , Endófitos/química , Pirrolidinonas/química , Antibacterianos/química , Antibacterianos/farmacología , Ascomicetos/genética , Bacterias/efectos de los fármacos , ADN de Hongos/genética , Ficus/microbiología , Genómica , Pruebas de Sensibilidad Microbiana , Estructura Molecular , FilogeniaRESUMEN
During the lytic phase of infection, the gamma herpesvirus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, 1.1 kb nuclear noncoding RNA of unknown function. We observe that this polyadenylated nuclear (PAN) RNA avidly binds host poly(A)-binding protein C1 (PABPC1), which normally functions in the cytoplasm to bind the poly(A) tails of mRNAs, regulating mRNA stability and translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of expression of the viral shutoff exonuclease (SOX) protein; SOX also mediates the host shutoff effect in which host mRNAs are downregulated while viral mRNAs are selectively expressed. We show that whereas PAN RNA is not required for the host shutoff effect or for PABPC1 re-localization, SOX strongly upregulates the levels of PAN RNA in transient transfection experiments. This upregulation is destroyed by the same SOX mutation that ablates the host shutoff effect and PABPC1 nuclear re-localization or by removal of the poly(A) tail of PAN. In cells induced into the KSHV lytic phase, depletion of PAN RNA using RNase H-targeting antisense oligonucleotides reveals that it is necessary for the production of late viral proteins from mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to long noncoding RNAs and suggest a mechanism of action for nuclear noncoding RNAs in gamma herpesvirus infection.