RESUMO
Serine is a vital amino acid in tumorigenesis. While cells can perform de novo serine synthesis, most transformed cells rely on serine uptake to meet their increased biosynthetic requirements. Solute carriers (SLCs), a family of transmembrane nutrient transport proteins, are the gatekeepers of amino acid acquisition and exchange in mammalian cells and are emerging as anticancer therapeutic targets; however, the SLCs that mediate serine transport in cancer cells remain unknown. Here we perform an arrayed RNAi screen of SLC-encoding genes while monitoring amino acid consumption and cell proliferation in colorectal cancer cells using metabolomics and high-throughput imaging. We identify SLC6A14 and SLC25A15 as major cytoplasmic and mitochondrial serine transporters, respectively. We also observe that SLC12A4 facilitates serine uptake. Dual targeting of SLC6A14 and either SLC25A15 or SLC12A4 diminishes serine uptake and growth of colorectal cancer cells in vitro and in vivo, particularly in cells with compromised de novo serine biosynthesis. Our results provide insight into the mechanisms that contribute to serine uptake and intracellular handling.
Assuntos
Neoplasias Colorretais , Proteínas de Membrana Transportadoras , Animais , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico , Aminoácidos/metabolismo , Serina/metabolismo , Neoplasias Colorretais/genética , Mamíferos/metabolismoRESUMO
BACKGROUND: Cellular metabolism is an integral component of cellular adaptation to stress, playing a pivotal role in the resistance of cancer cells to various treatment modalities, including radiotherapy. In response to radiotherapy, cancer cells engage antioxidant and DNA repair mechanisms which mitigate and remove DNA damage, facilitating cancer cell survival. Given the reliance of these resistance mechanisms on amino acid metabolism, we hypothesised that controlling the exogenous availability of the non-essential amino acids serine and glycine would radiosensitise cancer cells. METHODS: We exposed colorectal, breast and pancreatic cancer cell lines/organoids to radiation in vitro and in vivo in the presence and absence of exogenous serine and glycine. We performed phenotypic assays for DNA damage, cell cycle, ROS levels and cell death, combined with a high-resolution untargeted LCMS metabolomics and RNA-Seq. RESULTS: Serine and glycine restriction sensitised a range of cancer cell lines, patient-derived organoids and syngeneic mouse tumour models to radiotherapy. Comprehensive metabolomic and transcriptomic analysis of central carbon metabolism revealed that amino acid restriction impacted not only antioxidant response and nucleotide synthesis but had a marked inhibitory effect on the TCA cycle. CONCLUSION: Dietary restriction of serine and glycine is a viable radio-sensitisation strategy in cancer.
Assuntos
Neoplasias Pancreáticas , Serina , Camundongos , Animais , Serina/metabolismo , Glicina/farmacologia , Antioxidantes/metabolismo , AminoácidosRESUMO
Cancer cells have high demands for non-essential amino acids (NEAAs), which are precursors for anabolic and antioxidant pathways that support cell survival and proliferation. It is well-established that cancer cells consume the NEAA cysteine, and that cysteine deprivation can induce cell death; however, the specific factors governing acute sensitivity to cysteine starvation are poorly characterized. Here, we show that that neither expression of enzymes for cysteine synthesis nor availability of the primary precursor methionine correlated with acute sensitivity to cysteine starvation. We observed a strong correlation between efflux of the methionine-derived metabolite methylthioadenosine (MTA) and sensitivity to cysteine starvation. MTA efflux results from genetic deletion of methylthioadenosine phosphorylase (MTAP), which is frequently deleted in cancers. We show that MTAP loss upregulates polyamine metabolism which, concurrently with cysteine withdrawal, promotes elevated reactive oxygen species and prevents cell survival. Our results reveal an unexplored metabolic weakness at the intersection of polyamine and cysteine metabolism.
Assuntos
Cisteína/metabolismo , Redes e Vias Metabólicas , Neoplasias/metabolismo , Poliaminas/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Cisteína/deficiência , Feminino , Técnicas de Inativação de Genes , Humanos , Metionina/metabolismo , Camundongos , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Espécies Reativas de Oxigênio , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tracing the fate of carbon-13 (13C) labeled metabolites within cells by liquid chromatography mass spectrometry (LCMS) is a powerful analytical technique used for many years in the study of cell metabolism. Conventional experiments using LCMS and labeled nutrients tend to track the incorporation of 13C from exogenous nutrients (such as amino acids) into other, relatively proximal, cellular metabolites. Several labs have extended this technique to track transfer of 13C from the metabolite pool onto macromolecules, such as DNA, where methylation acts as an important functional modification. Here we describe a complete method that integrates previously established techniques to simultaneously track the use of 13C-serine or 13C-methionine into metabolite pools of the methionine cycle and into methylation of DNA and RNA. Given the ability to track methyl-transfer in a time-dependent way, this technique can provide temporal information about active methyl-transfer as well as quantification of total DNA/RNA methylation levels.
Assuntos
Isótopos de Carbono/metabolismo , Epigênese Genética , Metionina/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Isótopos de Nitrogênio/metabolismo , Serina/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida , Metilação de DNA , Epigenômica/métodos , Humanos , Hidrólise , RNA/genética , RNA/metabolismo , Espectrometria de Massas em TandemRESUMO
Macroautophagy can regulate cell signalling and tumorigenesis via elusive molecular mechanisms. We establish a RAS mutant cancer cell model where the autophagy gene ATG5 is dispensable in A549 cells in vitro, yet promotes tumorigenesis in mice. ATG5 represses transcriptional activation by the TGFß-SMAD gene regulatory pathway. However, autophagy does not terminate cytosolic signal transduction by TGFß. Instead, we use proteomics to identify selective degradation of the signalling scaffold TRAF3. TRAF3 autophagy is driven by RAS and results in activation of the NF-κB family member RELB. We show that RELB represses TGFß target promoters independently of DNA binding at NF-κB recognition sequences, instead binding with SMAD family member(s) at SMAD-response elements. Thus, autophagy antagonises TGFß gene expression. Finally, autophagy-deficient A549 cells regain tumorigenicity upon SMAD4 knockdown. Thus, at least in this setting, a physiologic function for autophagic regulation of gene expression is tumour growth.
Assuntos
Autofagia/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Smad/genética , Fator 3 Associado a Receptor de TNF/genética , Fator de Transcrição RelB/genética , Células A549 , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Smad/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelB/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Transplante HeterólogoRESUMO
Folate metabolism functions to supply one-carbon units that are vital for a range of biochemical reactions. Cancer cells can utilise serine as a major source of one-carbon units, rendering them dependent upon extracellular serine uptake or de novo serine synthesis for maximal growth and proliferation. One-carbon units are required for the production of critical cellular components, such as nucleotides, which enable cancer cells to maintain high proliferate rates. Of recent interest, one-carbon metabolism contributes to the biosynthesis and recycling of functional metabolites, such as ATP, S-adenosyl-methionine (SAM), and NAD(P)H, with important downstream consequences for cancer cell survival. In this review, we describe recent advances in our understanding of the importance of one-carbon metabolism in cancer, focussing upon the routes through which cancer cells obtain and use one-carbon units.
Assuntos
Neoplasias/metabolismo , Serina/metabolismo , Animais , Carbono/metabolismo , Proliferação de Células/fisiologia , HumanosRESUMO
Cells require one-carbon units for nucleotide synthesis, methylation and reductive metabolism, and these pathways support the high proliferative rate of cancer cells. As such, anti-folates, drugs that target one-carbon metabolism, have long been used in the treatment of cancer. Amino acids, such as serine are a major one-carbon source, and cancer cells are particularly susceptible to deprivation of one-carbon units by serine restriction or inhibition of de novo serine synthesis. Recent work has also begun to decipher the specific pathways and sub-cellular compartments that are important for one-carbon metabolism in cancer cells. In this review we summarise the historical understanding of one-carbon metabolism in cancer, describe the recent findings regarding the generation and usage of one-carbon units and explore possible future therapeutics that could exploit the dependency of cancer cells on one-carbon metabolism.
Assuntos
Aminoácidos/metabolismo , Carbono/metabolismo , Redes e Vias Metabólicas , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Humanos , Metilação , NAD/biossíntese , NADP/biossíntese , Nucleotídeos/biossínteseRESUMO
K-Ras dependent non-small cell lung cancer (NSCLC) cells are 'addicted' to basal autophagy that reprograms cellular metabolism in a lysosomal-sensitive manner. Here we demonstrate that the xenophagy-associated kinase TBK1 drives basal autophagy, consistent with its known requirement in K-Ras-dependent NSCLC proliferation. Furthermore, basal autophagy in this context is characterised by sequestration of the xenophagy cargo receptor Ndp52 and its paralogue Tax1bp1, which we demonstrate here to be a bona fide cargo receptor. Autophagy of these cargo receptors promotes non-canonical NF-κB signalling. We propose that this TBK1-dependent mechanism for NF-κB signalling contributes to autophagy addiction in K-Ras driven NSCLC.
Assuntos
Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Autofagia/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lentivirus , Neoplasias Pulmonares/genética , NF-kappa B/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismoRESUMO
Disrupted in schizophrenia 1 (DISC1) is a leading candidate susceptibility gene for schizophrenia, bipolar disorder and recurrent major depression, which has been implicated in other psychiatric illnesses of neurodevelopmental origin, including autism. DISC1 was initially identified at the breakpoint of a balanced chromosomal translocation, t(1;11) (q42.1;14.3), in a family with a high incidence of psychiatric illness. Carriers of the translocation show a 50% reduction in DISC1 protein levels, suggesting altered DISC1 expression as a pathogenic mechanism in psychiatric illness. Altered DISC1 expression in the post-mortem brains of individuals with psychiatric illness and the frequent implication of non-coding regions of the gene by association analysis further support this assertion. Here, we provide the first characterization of the DISC1 promoter region. Using dual luciferase assays, we demonstrate that a region -300 to -177 bp relative to the transcription start site (TSS) contributes positively to DISC1 promoter activity, while a region -982 to -301 bp relative to the TSS confers a repressive effect. We further demonstrate inhibition of DISC1 promoter activity and protein expression by forkhead-box P2 (FOXP2), a transcription factor implicated in speech and language function. This inhibition is diminished by two distinct FOXP2 point mutations, R553H and R328X, which were previously found in families affected by developmental verbal dyspraxia. Our work identifies an intriguing mechanistic link between neurodevelopmental disorders that have traditionally been viewed as diagnostically distinct but which do share varying degrees of phenotypic overlap.