Non-conserved metabolic regulation by LKB1 distinguishes human and mouse lung adenocarcinoma

KRAS is the most frequently mutated oncogene in human lung adenocarcinomas (hLUAD) and activating mutations in KRAS frequently co-occur with loss-of-function mutations in the tumor suppressor genes, TP53 or STK11/LKB1. However, mutation of all three genes is rarely observed in hLUAD, even though engineered mutations of all three genes produces a highly aggressive lung adenocarcinoma in mice (mLUAD). Here we provide an explanation of this difference between hLUAD and mLUAD by uncovering an evolutionary divergence in regulation of the glycolytic enzyme triosephosphate isomerase (TPI1). Using KRAS/TP53 mutant hLUAD cell lines, we show that TPI1 enzymatic activity can be altered via phosphorylation at Ser21 by the Salt Inducible Kinases (SIKs) in an LKB1-dependent manner; this allows modulation of glycolytic flux between completion of glycolysis and production of glycerol lipids. This metabolic flexibility appears to be critical in rapidly growing cells with KRAS and TP53 mutations, explaining why loss of LKB1 creates a metabolic liability in these tumors. In mice, the amino acid at position 21 of TPI1 is a Cys residue which can be oxidized to alter TPI1 activity, allowing regulation of glycolytic flux balance without a need for SIK kinases or LKB1. Our findings reveal an unexpected role for TPI1 in metabolic reprogramming and suggest that LKB1 and SIK family kinases are potential targets for treating KRAS/TP53 mutant hLUAD. Our data also provide a cautionary example of the limits of genetically engineered murine models as tools to study human diseases such as cancers.

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β-Hydroxybutyrate Oxidation Promotes the Accumulation of Immunometabolites in Activated Microglia Cells

Metabolites ◽

10.3390/metabo10090346 ◽

2020 ◽

Vol 10 (9) ◽

pp. 346

Author(s):

Adrian Benito ◽

Nabil Hajji ◽

Kevin O’Neill ◽

Hector C. Keun ◽

Nelofer Syed

Keyword(s):

Metabolic Regulation ◽

Marker Gene ◽

Tca Cycle ◽

Metabolic Reprogramming ◽

Glycolytic Flux ◽

Dihydroxyacetone Phosphate ◽

Glycolytic Intermediate ◽

Critical Set ◽

The Tca Cycle ◽

Bv2 Cells

Metabolic regulation of immune cells has arisen as a critical set of processes required for appropriate response to immunological signals. While our knowledge in this area has rapidly expanded in leukocytes, much less is known about the metabolic regulation of brain-resident microglia. In particular, the role of alternative nutrients to glucose remains poorly understood. Here, we use stable-isotope (13C) tracing strategies and metabolomics to characterize the oxidative metabolism of β-hydroxybutyrate (BHB) in human (HMC3) and murine (BV2) microglia cells and the interplay with glucose in resting and LPS-activated BV2 cells. We found that BHB is imported and oxidised in the TCA cycle in both cell lines with a subsequent increase in the cytosolic NADH:NAD+ ratio. In BV2 cells, stimulation with LPS upregulated the glycolytic flux, increased the cytosolic NADH:NAD+ ratio and promoted the accumulation of the glycolytic intermediate dihydroxyacetone phosphate (DHAP). The addition of BHB enhanced LPS-induced accumulation of DHAP and promoted glucose-derived lactate export. BHB also synergistically increased LPS-induced accumulation of succinate and other key immunometabolites, such as α-ketoglutarate and fumarate generated by the TCA cycle. Finally, BHB upregulated the expression of a key pro-inflammatory (M1 polarisation) marker gene, NOS2, in BV2 cells activated with LPS. In conclusion, we identify BHB as a potentially immunomodulatory metabolic substrate for microglia that promotes metabolic reprogramming during pro-inflammatory response.

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Abstract 261: Evaluating MTCH2 as a Modifier of Cardiomyopathy

Circulation Research ◽

10.1161/res.127.suppl_1.261 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Julie A Fischer ◽

Mattia Quattrocelli ◽

Megan Puckelwartz ◽

Matthew Wolf ◽

Elizabeth M McNally

Keyword(s):

Heart Failure ◽

Genetic Variation ◽

Oxygen Consumption ◽

Metabolic Regulation ◽

Critical Role ◽

Genetic Modifier ◽

Significant Risk ◽

Dependent Manner ◽

Loss Of Function ◽

Heart Tube

Background: Cardiomyopathy is a highly heritable disorder that carries a significant risk for heart failure and arrhythmias. The onset, severity, and progression of cardiomyopathy is influenced by genetic variation, with the most common form of inheritance in familial cases being autosomal dominant. All inherited cardiomyopathies are characterized by variable penetrance and expressivity, which in part arises from additional genetic variation, known as genetic modifiers. Methods and Results: Broad genomic profiling of human cardiomyopathy cases identified enriched genetic variation in the gene MTCH2. Specifically, a loss of function variant was found to be enriched in patients with cardiomyopathy. MTCH2 single nucleotide polymorphisms have also been linked to obesity, underscoring a critical role for MTCH2 in metabolic regulation. MTCH2 encodes a mitochondrial carrier protein that has a role in regulating oxidative phosphorylation. In order to investigate fundamental mechanisms by which MTCH2 contributes to cardiac and metabolic phenotypes, we generated a knockdown model of the Drosophila MTCH2 ortholog, Mtch. This knockdown model mimics what is seen in human carriers with the heterozygous loss of function allele. In the Drosophila model, Mtch RNA was reduced by approximately half. We found that cardiac-specific Mtch deficiency in flies produced heart tube dilated and reduced function as well as a shortened life span, documenting a clear role for cardiac Mtch. Cardiac deficiency of Mtch increased circulating lactate levels in flies. Oxygen consumption was reduced in cardiac Mtch deficiency flies in the presence of glucose, but not palmitate. Thus, loss of Mtch2 alters oxygen consumption in a substrate dependent manner. Conclusions: We identified MTCH2 as a modifier of the cardiomyopathy phenotype in humans. Reduction of Mtch in flies resulted in impaired cardiac function and reduced oxygen consumption under certain metabolic condition. As failed hearts are more dependent on glycolysis, these data support that reduction of MTCH2 promotes heart failure and provides a mechanism by which MTCH2 acts as a deleterious genetic modifier in heart failure.

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SMARCA4 supports the oncogenic landscape of KRAS-driven lung tumors

10.1101/2020.04.18.043927 ◽

2020 ◽

Author(s):

Shivani Malik ◽

Masaya Oshima ◽

Nilotpal Roy ◽

Swati Kaushik ◽

Ora Kuvshinova ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

High Frequency ◽

Lung Tumors ◽

Model Systems ◽

Mouse Lung ◽

Loss Of Function ◽

Recurrent Mutations ◽

Cancer Types ◽

And Function ◽

Lung Adenocarcinomas

AbstractCancer resequencing studies identify recurrent mutations in the switch/sucrose non-fermentable (SWI/SNF) complex at an unexpectedly high frequency across many cancer types. Some SWI/SNF mutations appear to be loss-of-function events, implying that the intact SWI/SNF complex is tumor suppressive. We examined the distribution and function of SMARCA4 mutations, the most frequently mutated SWI/SNF complex gene in lung adenocarcinoma, using human cancers, cell lines and mouse model systems. We found that lung adenocarcinomas harboring activated oncogenes have fewer deleterious mutations in SMARCA4 and express higher levels of the mRNA than cancers without activated oncogenes, indicating distinct dependencies on SMARCA4 in these two settings. Surprisingly, intact Smarca4 promoted the growth and tumorgenicity of KrasG12D-driven mouse lung tumors and human cells. Mechanistically, we found that Smarca4 supports the oncogenic transcriptional/signaling landscape of KrasG12D-driven mouse lung cancer. This dependency on the chromatin maintenance machinery in established cancer cells support treatments directed towards pathogenic SWI/SNF complexes in lung adenocarcinoma and other malignancies.

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Loss of Function of the Gene Encoding the Histone Methyltransferase KMT2D Leads to Deregulation of Mitochondrial Respiration

Cells ◽

10.3390/cells9071685 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1685

Author(s):

Consiglia Pacelli ◽

Iolanda Adipietro ◽

Natascia Malerba ◽

Gabriella Maria Squeo ◽

Claudia Piccoli ◽

...

Keyword(s):

Mitochondrial Respiratory Chain ◽

Significant Inhibition ◽

Metabolic Reprogramming ◽

Mass Spectrometry Analysis ◽

Metabolic Phenotype ◽

Glycolytic Flux ◽

Epigenetic Mark ◽

Loss Of Function ◽

Respiratory Chain Complexes ◽

Spectrometry Analysis

KMT2D encodes a methyltransferase responsible for histone 3 lysine 4 (H3K4) mono-/di-methylation, an epigenetic mark correlated with active transcription. Here, we tested the hypothesis that KMT2D pathogenic loss-of-function variants, which causes the Kabuki syndrome type 1, could affect the mitochondrial metabolic profile. By using Seahorse technology, we showed a significant reduction of the mitochondrial oxygen consumption rate as well as a reduction of the glycolytic flux in both Kmt2d knockout MEFs and skin fibroblasts of Kabuki patients harboring heterozygous KMT2D pathogenic variants. Mass-spectrometry analysis of intermediate metabolites confirmed alterations in the glycolytic and TCA cycle pathways. The observed metabolic phenotype was accompanied by a significant increase in the production of reactive oxygen species. Measurements of the specific activities of the mitochondrial respiratory chain complexes revealed significant inhibition of CI (NADH dehydrogenase) and CIV (cytochrome c oxidase); this result was further supported by a decrease in the protein content of both complexes. Finally, we unveiled an impaired oxidation of glucose and larger reliance on long-chain fatty acids oxidation. Altogether, our findings clearly indicate a rewiring of the mitochondrial metabolic phenotype in the KMT2D-null or loss-of-function context that might contribute to the development of Kabuki disease, and represents metabolic reprogramming as a potential new therapeutic approach.

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SMARCA4 deficiency-associated heterochromatin induces intrinsic DNA replication stress and susceptibility to ATR inhibition in lung adenocarcinoma

NAR Cancer ◽

10.1093/narcan/zcaa005 ◽

2020 ◽

Vol 2 (2) ◽

Author(s):

Kiminori Kurashima ◽

Hideto Kashiwagi ◽

Iwao Shimomura ◽

Ayako Suzuki ◽

Fumitaka Takeshita ◽

...

Keyword(s):

Dna Replication ◽

Lung Adenocarcinoma ◽

Human Cancer ◽

Replication Stress ◽

Dependent Manner ◽

Loss Of Function ◽

Heterochromatin Formation ◽

Dna Replication Stress

Abstract The SWI/SNF chromatin remodeling complex regulates transcription through the control of chromatin structure and is increasingly thought to play an important role in human cancer. Lung adenocarcinoma (LADC) patients frequently harbor mutations in SMARCA4, a core component of this multisubunit complex. Most of these mutations are loss-of-function mutations, which disrupt critical functions in the regulation of chromatin architecture and can cause DNA replication stress. This study reports that LADC cells deficient in SMARCA4 showed increased DNA replication stress and greater sensitivity to the ATR inhibitor (ATRi) in vitro and in vivo. Mechanistically, loss of SMARCA4 increased heterochromatin formation, resulting in stalled forks, a typical DNA replication stress. In the absence of SMARCA4, severe ATRi-induced single-stranded DNA, which caused replication catastrophe, was generated on nascent DNA near the reversed forks around heterochromatin in an Mre11-dependent manner. Thus, loss of SMARCA4 confers susceptibility to ATRi, both by increasing heterochromatin-associated replication stress and by allowing Mre11 to destabilize reversed forks. These two mechanisms synergistically increase susceptibility of SMARCA4-deficient LADC cells to ATRi. These results provide a preclinical basis for assessing SMARCA4 defects as a biomarker of ATRi efficacy.

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Noninvasive lung cancer detection via pulmonary protease profiling

10.1101/495259 ◽

2018 ◽

Cited By ~ 1

Author(s):

Jesse Kirkpatrick ◽

Andrew D. Warren ◽

Tuomas Tammela ◽

Peter M. K. Westcott ◽

Justin C. Voog ◽

...

Keyword(s):

Lung Cancer ◽

Mouse Model ◽

Lung Adenocarcinoma ◽

Early Stage ◽

Genetically Engineered ◽

Mouse Lung ◽

National Guidelines ◽

Early Stage Lung Cancer ◽

Early Detection Of Disease ◽

Genetically Engineered Mouse Model

AbstractLung cancer is the leading cause of cancer-related death and patients most commonly present with incurable metastatic disease. National guidelines recommend screening for high-risk patients with low-dose computed tomography (LDCT), but this approach has limitations including high false positive rates. Activity-based nanosensors (ABNs) detect dysregulated proteases in vivo and release a reporter to provide a urinary readout of disease activity. Here, we demonstrate the translational potential of ABNs by coupling ABN multiplexing with intrapulmonary delivery to detect early-stage lung cancer in an immunocompetent, genetically engineered mouse model (GEMM). The design of the multiplexed panel of sensors was informed by comparative transcriptomic analysis of human and mouse lung adenocarcinoma data sets and in vitro cleavage assays with recombinant candidate proteases. When employed in a Kras and Trp53 mutant lung adenocarcinoma mouse model, this approach confirmed the role of metalloproteases in lung cancer and enabled accurate early detection of disease, with 92% sensitivity and 100% specificity.

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Urinary detection of lung cancer in mice via noninvasive pulmonary protease profiling

Science Translational Medicine ◽

10.1126/scitranslmed.aaw0262 ◽

2020 ◽

Vol 12 (537) ◽

pp. eaaw0262 ◽

Cited By ~ 7

Author(s):

Jesse D. Kirkpatrick ◽

Andrew D. Warren ◽

Ava P. Soleimany ◽

Peter M. K. Westcott ◽

Justin C. Voog ◽

...

Keyword(s):

Lung Cancer ◽

Lung Adenocarcinoma ◽

Genetically Engineered ◽

Mouse Lung ◽

National Guidelines ◽

Stage Disease ◽

Risk Patients ◽

Localized Disease

Lung cancer is the leading cause of cancer-related death, and patients most commonly present with incurable advanced-stage disease. U.S. national guidelines recommend screening for high-risk patients with low-dose computed tomography, but this approach has limitations including high false-positive rates. Activity-based nanosensors can detect dysregulated proteases in vivo and release a reporter to provide a urinary readout of disease activity. Here, we demonstrate the translational potential of activity-based nanosensors for lung cancer by coupling nanosensor multiplexing with intrapulmonary delivery and machine learning to detect localized disease in two immunocompetent genetically engineered mouse models. The design of our multiplexed panel of sensors was informed by comparative transcriptomic analysis of human and mouse lung adenocarcinoma datasets and in vitro cleavage assays with recombinant candidate proteases. Intrapulmonary administration of the nanosensors to a Kras- and Trp53-mutant lung adenocarcinoma mouse model confirmed the role of metalloproteases in lung cancer and enabled accurate detection of localized disease, with 100% specificity and 81% sensitivity. Furthermore, this approach generalized to an alternative autochthonous model of lung adenocarcinoma, where it detected cancer with 100% specificity and 95% sensitivity and was not confounded by lipopolysaccharide-driven lung inflammation. These results encourage the clinical development of activity-based nanosensors for the detection of lung cancer.

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Matrix Stiffness Modulates Metabolic Interaction between Human Stromal and Breast Cancer Cells to Stimulate Epithelial Motility

Metabolites ◽

10.3390/metabo11070432 ◽

2021 ◽

Vol 11 (7) ◽

pp. 432

Author(s):

Iván Ponce ◽

Nelson Garrido ◽

Nicolás Tobar ◽

Francisco Melo ◽

Patricio C. Smith ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Cancer Progression ◽

Stromal Cells ◽

Glucose Transporter ◽

Lactate Production ◽

Resonance Energy ◽

Metabolic Reprogramming ◽

Dependent Manner ◽

Functional Link

Breast tumors belong to the type of desmoplastic lesion in which a stiffer tissue structure is a determinant of breast cancer progression and constitutes a risk factor for breast cancer development. It has been proposed that cancer-associated stromal cells (responsible for this fibrotic phenomenon) are able to metabolize glucose via lactate production, which supports the catabolic metabolism of cancer cells. The aim of this work was to investigate the possible functional link between these two processes. To measure the effect of matrix rigidity on metabolic determinations, we used compliant elastic polyacrylamide gels as a substrate material, to which matrix molecules were covalently linked. We evaluated metabolite transport in stromal cells using two different FRET (Fluorescence Resonance Energy Transfer) nanosensors specific for glucose and lactate. Cell migration/invasion was evaluated using Transwell devices. We show that increased stiffness stimulates lactate production and glucose uptake by mammary fibroblasts. This response was correlated with the expression of stromal glucose transporter Glut1 and monocarboxylate transporters MCT4. Moreover, mammary stromal cells cultured on stiff matrices generated soluble factors that stimulated epithelial breast migration in a stiffness-dependent manner. Using a normal breast stromal cell line, we found that a stiffer extracellular matrix favors the acquisition mechanistical properties that promote metabolic reprograming and also constitute a stimulus for epithelial motility. This new knowledge will help us to better understand the complex relationship between fibrosis, metabolic reprogramming, and cancer malignancy.

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Mitochondrial arginase-2 is essential for IL-10 metabolic reprogramming of inflammatory macrophages

Nature Communications ◽

10.1038/s41467-021-21617-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jennifer K. Dowling ◽

Remsha Afzal ◽

Linden J. Gearing ◽

Mariana P. Cervantes-Silva ◽

Stephanie Annett ◽

...

Keyword(s):

Metabolic Regulation ◽

Mitochondrial Dynamics ◽

Interleukin 10 ◽

Metabolic Reprogramming ◽

In Vivo Model ◽

Macrophage Polarisation ◽

Inflammatory Status ◽

Inflammatory Macrophages

AbstractMitochondria are important regulators of macrophage polarisation. Here, we show that arginase-2 (Arg2) is a microRNA-155 (miR-155) and interleukin-10 (IL-10) regulated protein localized at the mitochondria in inflammatory macrophages, and is critical for IL-10-induced modulation of mitochondrial dynamics and oxidative respiration. Mechanistically, the catalytic activity and presence of Arg2 at the mitochondria is crucial for oxidative phosphorylation. We further show that Arg2 mediates this process by increasing the activity of complex II (succinate dehydrogenase). Moreover, Arg2 is essential for IL-10-mediated downregulation of the inflammatory mediators succinate, hypoxia inducible factor 1α (HIF-1α) and IL-1β in vitro. Accordingly, HIF-1α and IL-1β are highly expressed in an LPS-induced in vivo model of acute inflammation using Arg2−/− mice. These findings shed light on a new arm of IL-10-mediated metabolic regulation, working to resolve the inflammatory status of the cell.

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Activation of mitochondrial TUFM ameliorates metabolic dysregulation through coordinating autophagy induction

Communications Biology ◽

10.1038/s42003-020-01566-0 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Dasol Kim ◽

Hui-Yun Hwang ◽

Eun Sun Ji ◽

Jin Young Kim ◽

Jong Shin Yoo ◽

...

Keyword(s):

Metabolic Regulation ◽

Elongation Factor ◽

Dependent Manner ◽

Diet Induced Obesity ◽

Metabolic Dysregulation ◽

Autophagy Induction ◽

Transcription Factor Eb ◽

Mitochondrial Elongation

AbstractDisorders of autophagy, a key regulator of cellular homeostasis, cause a number of human diseases. Due to the role of autophagy in metabolic dysregulation, there is a need to identify autophagy regulators as therapeutic targets. To address this need, we conducted an autophagy phenotype-based screen and identified the natural compound kaempferide (Kaem) as an autophagy enhancer. Kaem promoted autophagy through translocation of transcription factor EB (TFEB) without MTOR perturbation, suggesting it is safe for administration. Moreover, Kaem accelerated lipid droplet degradation in a lysosomal activity-dependent manner in vitro and ameliorated metabolic dysregulation in a diet-induced obesity mouse model. To elucidate the mechanism underlying Kaem’s biological activity, the target protein was identified via combined drug affinity responsive target stability and LC–MS/MS analyses. Kaem directly interacted with the mitochondrial elongation factor TUFM, and TUFM absence reversed Kaem-induced autophagy and lipid degradation. Kaem also induced mitochondrial reactive oxygen species (mtROS) to sequentially promote lysosomal Ca2+ efflux, TFEB translocation and autophagy induction, suggesting a role of TUFM in mtROS regulation. Collectively, these results demonstrate that Kaem is a potential therapeutic candidate/chemical tool for treating metabolic dysregulation and reveal a role for TUFM in autophagy for metabolic regulation with lipid overload.

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