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1.
Nature ; 586(7827): 120-126, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32968282

RESUMO

The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood1-3. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ and TNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, these data expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs.


Assuntos
Genoma/genética , Genômica , Neoplasias/genética , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Evasão Tumoral/genética , Evasão Tumoral/imunologia , Animais , Autofagia , Linhagem Celular Tumoral , Feminino , Genes Neoplásicos/genética , Humanos , Interferon gama/imunologia , Masculino , Camundongos , NF-kappa B/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais
2.
Nature ; 536(7617): 479-83, 2016 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-27509858

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease characterized by an intense fibrotic stromal response and deregulated metabolism. The role of the stroma in PDAC biology is complex and it has been shown to play critical roles that differ depending on the biological context. The stromal reaction also impairs the vasculature, leading to a highly hypoxic, nutrient-poor environment. As such, these tumours must alter how they capture and use nutrients to support their metabolic needs. Here we show that stroma-associated pancreatic stellate cells (PSCs) are critical for PDAC metabolism through the secretion of non-essential amino acids (NEAA). Specifically, we uncover a previously undescribed role for alanine, which outcompetes glucose and glutamine-derived carbon in PDAC to fuel the tricarboxylic acid (TCA) cycle, and thus NEAA and lipid biosynthesis. This shift in fuel source decreases the tumour's dependence on glucose and serum-derived nutrients, which are limited in the pancreatic tumour microenvironment. Moreover, we demonstrate that alanine secretion by PSCs is dependent on PSC autophagy, a process that is stimulated by cancer cells. Thus, our results demonstrate a novel metabolic interaction between PSCs and cancer cells, in which PSC-derived alanine acts as an alternative carbon source. This finding highlights a previously unappreciated metabolic network within pancreatic tumours in which diverse fuel sources are used to promote growth in an austere tumour microenvironment.


Assuntos
Alanina/metabolismo , Autofagia , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Células Estreladas do Pâncreas/citologia , Células Estreladas do Pâncreas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Vias Biossintéticas , Carbono/metabolismo , Carcinoma Ductal Pancreático/patologia , Ciclo do Ácido Cítrico , Feminino , Glucose/metabolismo , Xenoenxertos , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias Pancreáticas/patologia , Microambiente Tumoral/fisiologia
3.
Proc Natl Acad Sci U S A ; 114(5): 1129-1134, 2017 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-28096419

RESUMO

A fibroinflammatory stromal reaction cooperates with oncogenic signaling to influence pancreatic ductal adenocarcinoma (PDAC) initiation, progression, and therapeutic outcome, yet the mechanistic underpinning of this crosstalk remains poorly understood. Here we show that stromal cues elicit an adaptive response in the cancer cell including the rapid mobilization of a transcriptional network implicated in accelerated growth, along with anabolic changes of an altered metabolome. The close overlap of stroma-induced changes in vitro with those previously shown to be regulated by oncogenic Kras in vivo suggests that oncogenic Kras signaling-a hallmark and key driver of PDAC-is contingent on stromal inputs. Mechanistically, stroma-activated cancer cells show widespread increases in histone acetylation at transcriptionally enhanced genes, implicating the PDAC epigenome as a presumptive point of convergence between these pathways and a potential therapeutic target. Notably, inhibition of the bromodomain and extraterminal (BET) family of epigenetic readers, and of Bromodomain-containing protein 2 (BRD2) in particular, blocks stroma-inducible transcriptional regulation in vitro and tumor progression in vivo. Our work suggests the existence of a molecular "AND-gate" such that tumor activation is the consequence of mutant Kras and stromal cues, providing insight into the role of the tumor microenvironment in the origin and treatment of Ras-driven tumors.


Assuntos
Carcinoma Ductal Pancreático/fisiopatologia , Fibroblastos/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Código das Histonas , Metaboloma , Neoplasias Pancreáticas/fisiopatologia , Células Estromais/fisiologia , Microambiente Tumoral/fisiologia , Acetilação , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Citocinas/metabolismo , Metabolismo Energético , Elementos Facilitadores Genéticos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/fisiologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/fisiologia , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Fatores de Transcrição , Células Tumorais Cultivadas
4.
Blood ; 128(15): 1913-1917, 2016 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-27488349

RESUMO

The congenital sideroblastic anemias (CSAs) are a heterogeneous group of inherited blood disorders characterized by pathological mitochondrial iron deposition in erythroid precursors. Each known cause has been attributed to a mutation in a protein associated with heme biosynthesis, iron-sulfur cluster biogenesis, mitochondrial translation, or a component of the mitochondrial respiratory chain. Here, we describe a recurring mutation, c.276_278del, p.F93del, in NDUFB11, a mitochondrial respiratory complex I-associated protein encoded on the X chromosome, in 5 males with a variably syndromic, normocytic CSA. The p.F93del mutation results in respiratory insufficiency and loss of complex I stability and activity in patient-derived fibroblasts. Targeted introduction of this allele into K562 erythroleukemia cells results in a proliferation defect with minimal effect on erythroid differentiation potential, suggesting the mechanism of anemia in this disorder.


Assuntos
Anemia Sideroblástica/genética , Sequência de Bases , Cromossomos Humanos X/genética , Complexo I de Transporte de Elétrons/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Deleção de Sequência , Adolescente , Adulto , Idoso , Anemia Sideroblástica/metabolismo , Anemia Sideroblástica/patologia , Criança , Pré-Escolar , Cromossomos Humanos X/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Humanos , Células K562 , Masculino , Pessoa de Meia-Idade
6.
J Exp Med ; 221(9)2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39150482

RESUMO

Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function. Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.


Assuntos
ATP Citrato (pro-S)-Liase , Acetatos , Acetilcoenzima A , Linfócitos T CD8-Positivos , Cromatina , Camundongos Endogâmicos C57BL , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Animais , Cromatina/metabolismo , Acetilcoenzima A/metabolismo , ATP Citrato (pro-S)-Liase/metabolismo , ATP Citrato (pro-S)-Liase/genética , Camundongos , Acetatos/metabolismo , Acetato-CoA Ligase/metabolismo , Acetato-CoA Ligase/genética , Acetilação , Camundongos Knockout , Citosol/metabolismo , Histonas/metabolismo
7.
Cancer Metab ; 8: 1, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31908776

RESUMO

BACKGROUND: Metabolic programs in cancer cells are influenced by genotype and the tissue of origin. We have previously shown that central carbon metabolism is rewired in pancreatic ductal adenocarcinoma (PDA) to support proliferation through a glutamate oxaloacetate transaminase 1 (GOT1)-dependent pathway. METHODS: We utilized a doxycycline-inducible shRNA-mediated strategy to knockdown GOT1 in PDA and colorectal cancer (CRC) cell lines and tumor models of similar genotype. These cells were analyzed for the ability to form colonies and tumors to test if tissue type impacted GOT1 dependence. Additionally, the ability of GOT1 to impact the response to chemo- and radiotherapy was assessed. Mechanistically, the associated specimens were examined using a combination of steady-state and stable isotope tracing metabolomics strategies and computational modeling. Statistics were calculated using GraphPad Prism 7. One-way ANOVA was performed for experiments comparing multiple groups with one changing variable. Student's t test (unpaired, two-tailed) was performed when comparing two groups to each other. Metabolomics data comparing three PDA and three CRC cell lines were analyzed by performing Student's t test (unpaired, two-tailed) between all PDA metabolites and CRC metabolites. RESULTS: While PDA exhibits profound growth inhibition upon GOT1 knockdown, we found CRC to be insensitive. In PDA, but not CRC, GOT1 inhibition disrupted glycolysis, nucleotide metabolism, and redox homeostasis. These insights were leveraged in PDA, where we demonstrate that radiotherapy potently enhanced the effect of GOT1 inhibition on tumor growth. CONCLUSIONS: Taken together, these results illustrate the role of tissue type in dictating metabolic dependencies and provide new insights for targeting metabolism to treat PDA.

8.
Nat Commun ; 8: 15965, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28671190

RESUMO

Pancreatic ductal adenocarcinoma is a notoriously difficult-to-treat cancer and patients are in need of novel therapies. We have shown previously that these tumours have altered metabolic requirements, making them highly reliant on a number of adaptations including a non-canonical glutamine (Gln) metabolic pathway and that inhibition of downstream components of Gln metabolism leads to a decrease in tumour growth. Here we test whether recently developed inhibitors of glutaminase (GLS), which mediates an early step in Gln metabolism, represent a viable therapeutic strategy. We show that despite marked early effects on in vitro proliferation caused by GLS inhibition, pancreatic cancer cells have adaptive metabolic networks that sustain proliferation in vitro and in vivo. We use an integrated metabolomic and proteomic platform to understand this adaptive response and thereby design rational combinatorial approaches. We demonstrate that pancreatic cancer metabolism is adaptive and that targeting Gln metabolism in combination with these adaptive responses may yield clinical benefits for patients.


Assuntos
Glutamina/metabolismo , Redes e Vias Metabólicas , Neoplasias Pancreáticas/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Glutaminase/genética , Glutaminase/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/fisiopatologia , Proteômica , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias Pancreáticas
9.
J Natl Cancer Inst ; 107(10)2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26293574

RESUMO

BACKGROUND: Huntingtin (HTT) is mutated in Huntington's disease but is ubiquitously expressed, and mutant HTT influences cancer progression. We investigated wild-type HTT function during breast cancer. METHODS: We analyzed HTT and ZO1 expression as well as the HTT phosphoserine 421-activated form (S421-P-HTT) in human breast cancer tissues by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. We performed in vitro migration and invasion assays as well as in vivo tail vein injections of the metastatic 4T1 cells in BALB/c mice (n = 11 per group). We analyzed tumor progression in knock-in mice with modified S421 crossed with the MMTV-PyVT mammary cancer model (at least n = 12 per group). Data were analyzed with unpaired t tests, analysis of variance, Pearson or Spearman correlation, and Mann Whitney or Kruskal-Wallis tests. All statistical tests were two-sided. RESULTS: Levels of HTT and of S421-P-HTT are abnormally low in poorly differentiated and metastatic human breast cancers. HTT expression is downregulated in invasive compared with in situ carcinoma (P < .001). In BALB/c mice, silencing of HTT promotes lung colonization by a metastatic mammary cancer cell line (P = .005) and S421-unphosphorylatable-HTT accelerates cancer progression. HTT interacts with ZO1 and regulates both its expression and its localization to tight junctions. In human breast tumors, the patterns of HTT and ZO1 expression are similar (Pearson correlation coefficient = 0.66, P < .001). CONCLUSIONS: HTT may inhibit breast tumor dissemination through maintenance of ZO1 at tight junctions. Downregulation of HTT transcript and protein levels is a prognostic factor for poor prognosis and metastasis development.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Inativação Gênica , Proteínas do Tecido Nervoso/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Animais , Neoplasias da Mama/patologia , Movimento Celular , Progressão da Doença , Regulação para Baixo , Feminino , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína Huntingtina , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Proteínas do Tecido Nervoso/genética , Fosforilação , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Células Tumorais Cultivadas
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