RESUMO
Cancer cells frequently alter their lipids to grow and adapt to their environment1-3. Despite the critical functions of lipid metabolism in membrane physiology, signalling and energy production, how specific lipids contribute to tumorigenesis remains incompletely understood. Here, using functional genomics and lipidomic approaches, we identified de novo sphingolipid synthesis as an essential pathway for cancer immune evasion. Synthesis of sphingolipids is surprisingly dispensable for cancer cell proliferation in culture or in immunodeficient mice but required for tumour growth in multiple syngeneic models. Blocking sphingolipid production in cancer cells enhances the anti-proliferative effects of natural killer and CD8+ T cells partly via interferon-γ (IFNγ) signalling. Mechanistically, depletion of glycosphingolipids increases surface levels of IFNγ receptor subunit 1 (IFNGR1), which mediates IFNγ-induced growth arrest and pro-inflammatory signalling. Finally, pharmacological inhibition of glycosphingolipid synthesis synergizes with checkpoint blockade therapy to enhance anti-tumour immune response. Altogether, our work identifies glycosphingolipids as necessary and limiting metabolites for cancer immune evasion.
RESUMO
Cancer cells rewire their metabolism and rely on endogenous antioxidants to mitigate lethal oxidative damage to lipids. However, the metabolic processes that modulate the response to lipid peroxidation are poorly defined. Using genetic screens, we compared metabolic genes essential for proliferation upon inhibition of cystine uptake or glutathione peroxidase-4 (GPX4). Interestingly, very few genes were commonly required under both conditions, suggesting that cystine limitation and GPX4 inhibition may impair proliferation via distinct mechanisms. Our screens also identify tetrahydrobiopterin (BH4) biosynthesis as an essential metabolic pathway upon GPX4 inhibition. Mechanistically, BH4 is a potent radical-trapping antioxidant that protects lipid membranes from autoxidation, alone and in synergy with vitamin E. Dihydrofolate reductase catalyzes the regeneration of BH4, and its inhibition by methotrexate synergizes with GPX4 inhibition. Altogether, our work identifies the mechanism by which BH4 acts as an endogenous antioxidant and provides a compendium of metabolic modifiers of lipid peroxidation.
Assuntos
Cistina/metabolismo , Ferroptose/genética , Regulação Neoplásica da Expressão Gênica , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Tetra-Hidrofolato Desidrogenase/genética , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Biopterinas/análogos & derivados , Biopterinas/farmacologia , Carbolinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cistina/antagonistas & inibidores , Relação Dose-Resposta a Droga , Ferroptose/efeitos dos fármacos , Antagonistas do Ácido Fólico/farmacologia , Perfilação da Expressão Gênica , Humanos , Células Jurkat , Peroxidação de Lipídeos/efeitos dos fármacos , Metotrexato/farmacologia , Estresse Oxidativo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/antagonistas & inibidores , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Piperazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tetra-Hidrofolato Desidrogenase/metabolismo , Vitamina E/farmacologiaRESUMO
Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half-life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis-related genes in primary human stellate cells. RNA sequencing analysis of TGF-ß1-activated LX-2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS-induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. In mice expressing human RXFP1 gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α-smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.-Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.
Assuntos
Intoxicação por Tetracloreto de Carbono/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores de Peptídeos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Intoxicação por Tetracloreto de Carbono/genética , Linhagem Celular Transformada , Proliferação de Células/genética , Citocinas/genética , Citocinas/metabolismo , Humanos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Camundongos , Camundongos Transgênicos , Organoides/metabolismo , Organoides/patologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Transdução de Sinais/genéticaRESUMO
How an optimal level of human dopamine D4 receptor (hD4R) is maintained in synaptic membranes is not known. We show here that hD4R is ubiquitinated in primary neurons. We go on to show that ubiquitin is attached to hD4R through isopeptide and ester bonds. When lysine (Lys) residues of the hD4R are substituted with arginine (Arg) residues, cellular hD4R protein levels increase. A synergistic effect on hD4R levels is noted when cytoplasmic serine (Ser) and threonine (Thr) residues are mutated. Chloroquine, an inhibitor of lysosomal degradation, did not have an effect on hD4R protein levels. However, treatment with bortezomib, an inhibitor of the 20S proteasome, caused a dose-dependent increase in hD4R protein levels. The effect of bortezomib was attenuated in the receptor variants that lacked Lys or Ser/Thr residues, and the hD4R mutant that lacked 17 cytoplasmic Lys, Ser, and Thr residues was nearly insensitive to bortezomib treatment. We conclude that both isopeptide and ester bond ubiquitination regulate proteasomal degradation of hD4R.
Assuntos
Receptores de Dopamina D4/metabolismo , Receptores de Dopamina D4/fisiologia , Sequência de Aminoácidos , Arginina , Bortezomib , Células HEK293 , Humanos , Lisina , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Receptores Dopaminérgicos , Receptores de Dopamina D4/química , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Stress-adaptive mechanisms enable tumour cells to overcome metabolic constraints under nutrient and oxygen shortage. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumour growth are poorly understood. Here we identify GOT2-catalysed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumour cells under hypoxic culture conditions. In contrast, GOT2-catalysed aspartate synthesis is dispensable for pancreatic tumour formation in vivo. The dependence of pancreatic tumour cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRAS dependent, and is mediated by hypoxia-inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9). Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRAS tumours can overcome nutrient deprivation under hypoxic conditions.
Assuntos
Ácido Aspártico , Neoplasias Pancreáticas , Linhagem Celular Tumoral , Humanos , Hipóxia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Diseases, such as diabetes and hypertension, often lead to chronic kidney failure. The peptide hormone relaxin has been shown to have therapeutic effects in various organs. In the present study, we tested the hypothesis that ML290, a small molecule agonist of the human relaxin receptor (RXFP1), is able to target the kidney to remodel the extracellular matrix and reduce apoptosis induced by unilateral ureteral obstruction (UUO). UUO was performed on the left kidney of humanized RXFP1 mice, where the right kidneys served as contralateral controls. Mice were randomly allocated to receive either vehicle or ML290 (30 mg/kg) via daily intraperitoneal injection, and kidneys were collected for apoptosis, RNA, and protein analyses. UUO significantly increased expression of pro-apoptotic markers in both vehicle- and ML290-treated mice when compared to their contralateral control kidneys. Specifically, Bax expression and Erk1/2 activity were upregulated, accompanied by an increase of TUNEL-positive cells in the UUO kidneys. Additionally, UUO induced marked increase in myofibroblast differentiation and aberrant remodeling on the extracellular matrix. ML290 suppressed these processes by promoting a reduction of pro-apoptotic, fibroblastic, and inflammatory markers in the UUO kidneys. Finally, the potent effects of ML290 to remodel the extracellular matrix were demonstrated by its ability to reduce collagen gene expression in the UUO kidneys. Our data indicate that daily administration of ML290 has renal protective effects in the UUO mouse model, specifically through its anti-apoptotic and extracellular matrix remodeling properties.
RESUMO
Relaxin, a small peptide hormone of the insulin/relaxin family, demonstrated antifibrotic, organ protective, vasodilatory, and proangiogenic properties in clinical trials and several animal models of human diseases. Relaxin family peptide receptor 1 (RXFP1) is the relaxin cognate G protein-coupled receptor. We have identified a series of small molecule agonists of human RXFP1. The lead compound ML290 demonstrated preferred absorption, distribution, metabolism, and excretion profiles, is easy to synthesize, and has high stability in vivo. However, ML290 does not activate rodent RXFP1s and therefore cannot be tested in common preclinical animal models. Here we describe the production and analysis of a mouse transgenic model, a knock-out/knock-in of the human RXFP1 (hRXFP1) complementary DNA into the mouse Rxfp1 (mRxfp1) gene. Insertion of the vector into the mRxfp1 locus caused disruption of mRxfp1 and expression of hRXFP1. The transcriptional expression pattern of the hRXFP1 allele was similar to mRxfp1. Female mice homozygous for hRXFP1 showed relaxation of the pubic symphysis at parturition and normal development of mammary nipples and vaginal epithelium, indicating full complementation of mRxfp1 gene ablation. Intravenous injection of relaxin led to an increase in heart rate in humanized and wild-type females but not in Rxfp1-deficient mice, whereas ML290 increased heart rate in humanized but not wild-type animals, suggesting specific target engagement by ML290. Moreover, intraperitoneal injection of ML290 caused a decrease in blood osmolality. Taken together, our data show humanized RXFP1 mice can be used for testing relaxin receptor modulators in various preclinical studies.