Lactate Metabolism in Human Lung Tumors.

Cancer cells consume glucose and secrete lactate in culture. It is unknown whether lactate contributes to energy metabolism in living tumors. We previously reported that human non-small-cell lung cancers (NSCLCs) oxidize glucose in the tricarboxylic acid (TCA) cycle. Here, we show that lactate is also a TCA cycle carbon source for NSCLC. In human NSCLC, evidence of lactate utilization was most apparent in tumors with high 18fluorodeoxyglucose uptake and aggressive oncological behavior. Infusing human NSCLC patients with 13C-lactate revealed extensive labeling of TCA cycle metabolites. In mice, deleting monocarboxylate transporter-1 (MCT1) from tumor cells eliminated lactate-dependent metabolite labeling, confirming tumor-cell-autonomous lactate uptake. Strikingly, directly comparing lactate and glucose metabolism in vivo indicated that lactate's contribution to the TCA cycle predominates. The data indicate that tumors, including bona fide human NSCLC, can use lactate as a fuel in vivo.

Compartmentalized metabolism supports midgestation mammalian development.

Mammalian embryogenesis requires rapid growth and proper metabolic regulation1. Midgestation features increasing oxygen and nutrient availability concomitant with fetal organ development2,3. Understanding how metabolism supports development requires approaches to observe metabolism directly in model organisms in utero. Here we used isotope tracing and metabolomics to identify evolving metabolic programmes in the placenta and embryo during midgestation in mice. These tissues differ metabolically throughout midgestation, but we pinpointed gestational days (GD) 10.5-11.5 as a transition period for both placenta and embryo. Isotope tracing revealed differences in carbohydrate metabolism between the tissues and rapid glucose-dependent purine synthesis, especially in the embryo. Glucose's contribution to the tricarboxylic acid (TCA) cycle rises throughout midgestation in the embryo but not in the placenta. By GD12.5, compartmentalized metabolic programmes are apparent within the embryo, including different nutrient contributions to the TCA cycle in different organs. To contextualize developmental anomalies associated with Mendelian metabolic defects, we analysed mice deficient in LIPT1, the enzyme that activates 2-ketoacid dehydrogenases related to the TCA cycle4,5. LIPT1 deficiency suppresses TCA cycle metabolism during the GD10.5-GD11.5 transition, perturbs brain, heart and erythrocyte development and leads to embryonic demise by GD11.5. These data document individualized metabolic programmes in developing organs in utero.

γ-6-Phosphogluconolactone, a Byproduct of the Oxidative Pentose Phosphate Pathway, Contributes to AMPK Activation through Inhibition of PP2A.

The oxidative pentose phosphate pathway (oxiPPP) contributes to cell metabolism through not only the production of metabolic intermediates and reductive NADPH but also inhibition of LKB1-AMPK signaling by ribulose-5-phosphate (Ru-5-P), the product of the third oxiPPP enzyme 6-phosphogluconate dehydrogenase (6PGD). However, we found that knockdown of glucose-6-phosphate dehydrogenase (G6PD), the first oxiPPP enzyme, did not affect AMPK activation despite decreased Ru-5-P and subsequent LKB1 activation, due to enhanced activity of PP2A, the upstream phosphatase of AMPK. In contrast, knockdown of 6PGD or 6-phosphogluconolactonase (PGLS), the second oxiPPP enzyme, reduced PP2A activity. Mechanistically, knockdown of G6PD or PGLS decreased or increased 6-phosphogluconolactone level, respectively, which enhanced the inhibitory phosphorylation of PP2A by Src. Furthermore, γ-6-phosphogluconolactone, an oxiPPP byproduct with unknown function generated through intramolecular rearrangement of δ-6-phosphogluconolactone, the only substrate of PGLS, bound to Src and enhanced PP2A recruitment. Together, oxiPPP regulates AMPK homeostasis by balancing the opposing LKB1 and PP2A.

SNAT7 regulates mTORC1 via macropinocytosis.

Mammalian target of rapamycin complex 1 (mTORC1) senses amino acids to control cell growth, metabolism, and autophagy. Some amino acids signal to mTORC1 through the Rag GTPase, whereas glutamine and asparagine activate mTORC1 through a Rag GTPase-independent pathway. Here, we show that the lysosomal glutamine and asparagine transporter SNAT7 activates mTORC1 after extracellular protein, such as albumin, is macropinocytosed. The N terminus of SNAT7 forms nutrient-sensitive interaction with mTORC1 and regulates mTORC1 activation independently of the Rag GTPases. Depletion of SNAT7 inhibits albumin-induced mTORC1 lysosomal localization and subsequent activation. Moreover, SNAT7 is essential to sustain KRAS-driven pancreatic cancer cell growth through mTORC1. Thus, SNAT7 links glutamine and asparagine signaling from extracellular protein to mTORC1 independently of the Rag GTPases and is required for macropinocytosis-mediated mTORC1 activation and pancreatic cancer cell growth.

The transcription factors aryl hydrocarbon receptor and MYC cooperate in the regulation of cellular metabolism.

The transcription factor AHR (aryl hydrocarbon receptor) drives the expression of genes involved in detoxification pathways in cells exposed to pollutants and other small molecules. Moreover, AHR supports transcriptional programs that promote ribosome biogenesis and protein synthesis in cells stimulated to proliferate by the oncoprotein MYC. Thus, AHR is necessary for the proliferation of MYC-overexpressing cells. To define metabolic pathways in which AHR cooperates with MYC in supporting cell growth, here we used LC-MS-based metabolomics to examine the metabolome of MYC-expressing cells upon AHR knockdown. We found that AHR knockdown reduced lactate, S-lactoylglutathione, N-acetyl-l-alanine, 2-hydroxyglutarate, and UMP levels. Using our previously obtained RNA sequencing data, we found that AHR mediates the expression of the UMP-generating enzymes dihydroorotate dehydrogenase (quinone) (DHODH) and uridine monophosphate synthetase (UMPS), as well as lactate dehydrogenase A (LDHA), establishing a mechanism by which AHR regulates lactate and UMP production in MYC-overexpressing cells. AHR knockdown in glioblastoma cells also reduced the expression of LDHA (and lactate), DHODH, and UMPS but did not affect UMP levels, likely because of compensatory mechanisms in these cells. Our results indicate that AHR contributes to the regulation of metabolic pathways necessary for the proliferation of transformed cells.

Induction of LEF1 by MYC activates the WNT pathway and maintains cell proliferation.

BACKGROUND: While regulated WNT activity is required for normal development and stem cell maintenance, mutations that lead to constitutive activation of the WNT pathway cause cellular transformation and drive colorectal cancer. Activation of the WNT pathway ultimately leads to the nuclear translocation of ß-catenin which, in complex with TCF/LEF factors, promotes the transcription of genes necessary for growth. The proto-oncogene MYC is one of the most critical genes activated downstream the WNT pathway in colon cancer. Here, we investigate the converse regulation of the WNT pathway by MYC. METHODS: We performed RNA-seq analyses to identify genes regulated in cells expressing MYC. We validated the regulation of genes in the WNT pathway including LEF1 by MYC using RT-qPCR, Western blotting, and ChIP-seq. We investigated the importance of LEF1 for the viability of MYC-expressing cells in in fibroblasts, epithelial cells, and colon cells. Bioinformatic analyses were utilized to define the expression of MYC-regulated genes in human colon cancer and metabolomics analyses were used to identify pathways regulated by LEF1 in MYC expressing cells. RESULTS: MYC regulates the levels of numerous WNT-related genes, including the ß-catenin co-transcription factor LEF1. MYC activates the transcription of LEF1 and is required for LEF1 expression in colon cancer cells and in primary colonic cells transformed by APC loss of function, a common mutation in colon cancer patients. LEF1 caused the retention of ß-catenin in the nucleus, leading to the activation of the WNT pathway in MYC-expressing cells. Consequently, MYC-expressing cells were sensitive to LEF1 inhibition. Moreover, we describe two examples of genes induced in MYC-expressing cells that require LEF1 activity: the peroxisome proliferator activated receptor delta (PPARδ) and the Acyl CoA dehydrogenase 9 (ACAD9). CONCLUSIONS: We demonstrated that MYC is a transcriptional regulator of LEF1 in colonic cells. Our work proposes a novel pathway by which MYC regulates proliferation through activating LEF1 expression which in turn activates the WNT pathway.

HILIC and ERLIC Enrichment of Glycopeptides Derived from Breast and Brain Cancer Cells.

Aberrant glycosylation has been linked to many different cancer types. The blood-brain barrier (BBB) is a region of the brain that regulates the entrance of ions, diseases, toxins, and so on. However, in breast cancer metastasis, the BBB fails to prevent the crossing of the cancer cells into the brain. Here we present a study of identifying and quantifying the glycosylation of six breast and brain cancer cell lines using hydrophilic interaction liquid chromatography (HILIC) and electrostatic repulsion liquid chromatography (ERLIC) enrichments and LC-MS/MS analysis. Qualitative and quantitative analyses of N-linked glycosylation were performed by both enrichment techniques for individual and complementary comparison. Potential cancer glycopeptide biomarkers were identified and confirmed by chemometric and statistical evaluations. A total of 497 glycopeptides were characterized, of which 401 were common glycopeptides (80.6% overlap) identified from both enrichment techniques. HILIC enrichment yielded 320 statistically significant glycopeptides in 231BR relative to the other cell lines out of 494 unique glycopeptides, and sequential HILIC-ERLIC enrichment yielded 214 statistically significant glycopeptides in 231BR compared with the other cell lines out of 404 unique glycopeptides. The results provide the first comprehensive glycopeptide listing for these six cell lines.

Automated Glycan Sequencing from Tandem Mass Spectra of N-Linked Glycopeptides.

Mass spectrometry has become a routine experimental tool for proteomic biomarker analysis of human blood samples, partly due to the large availability of informatics tools. As one of the most common protein post-translational modifications (PTMs) in mammals, protein glycosylation has been observed to alter in multiple human diseases and thus may potentially be candidate markers of disease progression. While mass spectrometry instrumentation has seen advancements in capabilities, discovering glycosylation-related markers using existing software is currently not straightforward. Complete characterization of protein glycosylation requires the identification of intact glycopeptides in samples, including identification of the modification site as well as the structure of the attached glycans. In this paper, we present GlycoSeq, an open-source software tool that implements a heuristic iterated glycan sequencing algorithm coupled with prior knowledge for automated elucidation of the glycan structure within a glycopeptide from its collision-induced dissociation tandem mass spectrum. GlycoSeq employs rules of glycosidic linkage as defined by glycan synthetic pathways to eliminate improbable glycan structures and build reasonable glycan trees. We tested the tool on two sets of tandem mass spectra of N-linked glycopeptides cell lines acquired from breast cancer patients. After employing enzymatic specificity within the N-linked glycan synthetic pathway, the sequencing results of GlycoSeq were highly consistent with the manually curated glycan structures. Hence, GlycoSeq is ready to be used for the characterization of glycan structures in glycopeptides from MS/MS analysis. GlycoSeq is released as open source software at https://github.com/chpaul/GlycoSeq/ .

Integrated Transcriptomic and Glycomic Profiling of Glioma Stem Cell Xenografts.

Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) have the innate ability to migrate or home toward and engraft in tumors such as glioblastoma (GBM). Because of this unique property of BM-hMSCs, we have explored their use for cell-mediated therapeutic delivery for the advancement of GBM treatment. Extravasation, the process by which blood-borne cells­such as BM-hMSCs­enter the tissue, is a highly complex process but is heavily dependent upon glycosylation for glycan-glycan and glycan-protein adhesion between the cell and endothelium. However, in a translationally significant preclinical glioma stem cell xenograft (GSCX) model of GBM, BM-hMSCs demonstrate unequal tropism toward these tumors. We hypothesized that there may be differences in the glycan compositions between the GSCXs that elicit homing ("attractors") and those that do not ("non-attractors") that facilitate or impede the engraftment of BM-hMSCs in the tumor. In this study, glycotranscriptomic analysis revealed significant heterogeneity within the attractor phenotype and the enrichment of high mannose type N-glycan biosynthesis in the non-attractor phenotype. Orthogonal validation with topical PNGase F deglycosylation on the tumor regions of xenograft tissue, followed by nLC-ESI-MS, confirmed the presence of increased high mannose type N-glycans in the non-attractors. Additional evidence provided by our glycomic study revealed the prevalence of terminal sialic acid-containing N-glycans in non-attractors and terminal galactose and N-acetyl-glucosamine N-glycans in attractors. Our results provide the first evidence for differential glycomic profiles in attractor and non-attractor GSCXs and extend the scope of molecular determinates in BM-hMSC homing to glioma.

Electron transport chain inhibition increases cellular dependence on purine transport and salvage.

Mitochondria house many metabolic pathways required for homeostasis and growth. To explore how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts from patients with various mitochondrial disorders and cancer cells with electron transport chain (ETC) blockade. These analyses revealed extensive perturbations in purine metabolism, and stable isotope tracing demonstrated that ETC defects suppress de novo purine synthesis while enhancing purine salvage. In human lung cancer, tumors with markers of low oxidative mitochondrial metabolism exhibit enhanced expression of the salvage enzyme hypoxanthine phosphoribosyl transferase 1 (HPRT1) and high levels of the HPRT1 product inosine monophosphate. Mechanistically, ETC blockade activates the pentose phosphate pathway, providing phosphoribosyl diphosphate to drive purine salvage supplied by uptake of extracellular bases. Blocking HPRT1 sensitizes cancer cells to ETC inhibition. These findings demonstrate how cells remodel purine metabolism upon ETC blockade and uncover a new metabolic vulnerability in tumors with low respiration.

Metabolic inflexibility promotes mitochondrial health during liver regeneration.

Mitochondria are critical for proper organ function and mechanisms to promote mitochondrial health during regeneration would benefit tissue homeostasis. We report that during liver regeneration, proliferation is suppressed in electron transport chain (ETC)-dysfunctional hepatocytes due to an inability to generate acetyl-CoA from peripheral fatty acids through mitochondrial ß-oxidation. Alternative modes for acetyl-CoA production from pyruvate or acetate are suppressed in the setting of ETC dysfunction. This metabolic inflexibility forces a dependence on ETC-functional mitochondria and restoring acetyl-CoA production from pyruvate is sufficient to allow ETC-dysfunctional hepatocytes to proliferate. We propose that metabolic inflexibility within hepatocytes can be advantageous by limiting the expansion of ETC-dysfunctional cells.

Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples.

Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C positions (isotopomers), whereas NMR is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring less than 1% of the sample used for NMR. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase (FH) deficiency in human renal cancers, and investigate the contributions of tricarboxylic acid (TCA) cycle turnover and CO2 recycling to isotope labeling in vivo. This method can accompany NMR or standard MS to provide outstanding sensitivity in isotope-labeling experiments, particularly in vivo.

Limiting mitochondrial plasticity by targeting DRP1 induces metabolic reprogramming and reduces breast cancer brain metastases.

Disseminated tumor cells with metabolic flexibility to utilize available nutrients in distal organs persist, but the precise mechanisms that facilitate metabolic adaptations remain unclear. Here we show fragmented mitochondrial puncta in latent brain metastatic (Lat) cells enable fatty acid oxidation (FAO) to sustain cellular bioenergetics and maintain redox homeostasis. Depleting the enriched dynamin-related protein 1 (DRP1) and limiting mitochondrial plasticity in Lat cells results in increased lipid droplet accumulation, impaired FAO and attenuated metastasis. Likewise, pharmacological inhibition of DRP1 using a small-molecule brain-permeable inhibitor attenuated metastatic burden in preclinical models. In agreement with these findings, increased phospho-DRP1 expression was observed in metachronous brain metastasis compared with patient-matched primary tumors. Overall, our findings reveal the pivotal role of mitochondrial plasticity in supporting the survival of Lat cells and highlight the therapeutic potential of targeting cellular plasticity programs in combination with tumor-specific alterations to prevent metastatic recurrences.

Electron transport chain inhibition increases cellular dependence on purine transport and salvage.

Cancer cells reprogram their metabolism to support cell growth and proliferation in harsh environments. While many studies have documented the importance of mitochondrial oxidative phosphorylation (OXPHOS) in tumor growth, some cancer cells experience conditions of reduced OXPHOS in vivo and induce alternative metabolic pathways to compensate. To assess how human cells respond to mitochondrial dysfunction, we performed metabolomics in fibroblasts and plasma from patients with inborn errors of mitochondrial metabolism, and in cancer cells subjected to inhibition of the electron transport chain (ETC). All these analyses revealed extensive perturbations in purine-related metabolites; in non-small cell lung cancer (NSCLC) cells, ETC blockade led to purine metabolite accumulation arising from a reduced cytosolic NAD + /NADH ratio (NADH reductive stress). Stable isotope tracing demonstrated that ETC deficiency suppressed de novo purine nucleotide synthesis while enhancing purine salvage. Analysis of NSCLC patients infused with [U- 13 C]glucose revealed that tumors with markers of low oxidative mitochondrial metabolism exhibited high expression of the purine salvage enzyme HPRT1 and abundant levels of the HPRT1 product inosine monophosphate (IMP). ETC blockade also induced production of ribose-5' phosphate (R5P) by the pentose phosphate pathway (PPP) and import of purine nucleobases. Blocking either HPRT1 or nucleoside transporters sensitized cancer cells to ETC inhibition, and overexpressing nucleoside transporters was sufficient to drive growth of NSCLC xenografts. Collectively, this study mechanistically delineates how cells compensate for suppressed purine metabolism in response to ETC blockade, and uncovers a new metabolic vulnerability in tumors experiencing NADH excess.

An interactive web application for exploring human plasma and fibroblast metabolomics data from patients with inborn errors of metabolism.

Metabolomic profiling is instrumental in understanding the systemic and cellular impact of inborn errors of metabolism (IEMs), monogenic disorders caused by pathogenic genomic variants in genes involved in metabolism. This study encompasses untargeted metabolomics analysis of plasma from 474 individuals and fibroblasts from 67 subjects, incorporating healthy controls, patients with 65 different monogenic diseases, and numerous undiagnosed cases. We introduce a web application designed for the in-depth exploration of this extensive metabolomics database. The application offers a user-friendly interface for data review, download, and detailed analysis of metabolic deviations linked to IEMs at the level of individual patients or groups of patients with the same diagnosis. It also provides interactive tools for investigating metabolic relationships and offers comparative analyses of plasma and fibroblast profiles. This tool emphasizes the metabolic interplay within and across biological matrices, enriching our understanding of metabolic regulation in health and disease. As a resource, the application provides broad utility in research, offering novel insights into metabolic pathways and their alterations in various disorders.

Optimized protocol for stable isotope tracing and steady-state metabolomics in mouse HER2+ breast cancer brain metastasis.

Analyzing the metabolic dependencies of tumor cells is vital for cancer diagnosis and treatment. Here, we describe a protocol for 13C-stable glucose and glutamine isotope tracing in mice HER2+ breast cancer brain metastatic lesions. We describe how to inject cancer cells intracardially to generate brain metastatic lesions in mice. We then detail how to perform 13C-stable isotope infusion in mice with established brain metastasis. Finally, we outline steps for sample collection, processing for metabolite extraction, and analyzing mass spectrometry data. For complete details on the use and execution of this protocol, please refer to Parida et al. (2022).

Principles of reproducible metabolite profiling of enriched lymphocytes in tumors and ascites from human ovarian cancer.

Identifying metabolites and delineating their immune-regulatory contribution in the tumor microenvironment is an area of intense study. Interrogating metabolites and metabolic networks among immune cell subsets and host cells from resected tissues and fluids of human patients presents a major challenge, owing to the specialized handling of samples for downstream metabolomics. To address this, we first outline the importance of collaborating with a biobank for coordinating and streamlining workflow for point of care, sample collection, processing and cryopreservation. After specimen collection, we describe our 60-min rapid bead-based cellular enrichment method that supports metabolite analysis between T cells and tumor cells by mass spectrometry. We also describe how the metabolic data can be complemented with metabolic profiling by flow cytometry. This protocol can serve as a foundation for interrogating the metabolism of cell subsets from primary human ovarian cancer.

In vivo isotope tracing reveals a requirement for the electron transport chain in glucose and glutamine metabolism by tumors.

In mice and humans with cancer, intravenous 13C-glucose infusion results in 13C labeling of tumor tricarboxylic acid (TCA) cycle intermediates, indicating that pyruvate oxidation in the TCA cycle occurs in tumors. The TCA cycle is usually coupled to the electron transport chain (ETC) because NADH generated by the cycle is reoxidized to NAD+ by the ETC. However, 13C labeling does not directly report ETC activity, and other pathways can oxidize NADH, so the ETC's role in these labeling patterns is unverified. We examined the impact of the ETC complex I inhibitor IACS-010759 on tumor 13C labeling. IACS-010759 suppresses TCA cycle labeling from glucose or lactate and increases labeling from glutamine. Cancer cells expressing yeast NADH dehydrogenase-1, which recycles NADH to NAD+ independently of complex I, display normalized labeling when complex I is inhibited, indicating that cancer cell ETC activity regulates TCA cycle metabolism and 13C labeling from multiple nutrients.

In vivo characterization of glutamine metabolism identifies therapeutic targets in clear cell renal cell carcinoma.

Targeting metabolic vulnerabilities has been proposed as a therapeutic strategy in renal cell carcinoma (RCC). Here, we analyzed the metabolism of patient-derived xenografts (tumorgrafts) from diverse subtypes of RCC. Tumorgrafts from VHL-mutant clear cell RCC (ccRCC) retained metabolic features of human ccRCC and engaged in oxidative and reductive glutamine metabolism. Genetic silencing of isocitrate dehydrogenase-1 or isocitrate dehydrogenase-2 impaired reductive labeling of tricarboxylic acid (TCA) cycle intermediates in vivo and suppressed growth of tumors generated from tumorgraft-derived cells. Glutaminase inhibition reduced the contribution of glutamine to the TCA cycle and resulted in modest suppression of tumorgraft growth. Infusions with [amide-15N]glutamine revealed persistent amidotransferase activity during glutaminase inhibition, and blocking these activities with the amidotransferase inhibitor JHU-083 also reduced tumor growth in both immunocompromised and immunocompetent mice. We conclude that ccRCC tumorgrafts catabolize glutamine via multiple pathways, perhaps explaining why it has been challenging to achieve therapeutic responses in patients by inhibiting glutaminase.

De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma.

Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH's ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.