Spatial single-cell isotope tracing reveals heterogeneity of de novo fatty acid synthesis in cancer.

While heterogeneity is a key feature of cancer, understanding metabolic heterogeneity at the single-cell level remains a challenge. Here we present 13C-SpaceM, a method for spatial single-cell isotope tracing that extends the previously published SpaceM method with detection of 13C6-glucose-derived carbons in esterified fatty acids. We validated 13C-SpaceM on spatially heterogeneous models using liver cancer cells subjected to either normoxia-hypoxia or ATP citrate lyase depletion. This revealed substantial single-cell heterogeneity in labelling of the lipogenic acetyl-CoA pool and in relative fatty acid uptake versus synthesis hidden in bulk analyses. Analysing tumour-bearing brain tissue from mice fed a 13C6-glucose-containing diet, we found higher glucose-dependent synthesis of saturated fatty acids and increased elongation of essential fatty acids in tumours compared with healthy brains. Furthermore, our analysis uncovered spatial heterogeneity in lipogenic acetyl-CoA pool labelling in tumours. Our method enhances spatial probing of metabolic activities in single cells and tissues, providing insights into fatty acid metabolism in homoeostasis and disease.

mTORC1 regulates cell survival under glucose starvation through 4EBP1/2-mediated translational reprogramming of fatty acid metabolism.

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells.

13C-SpaceM: Spatial single-cell isotope tracing reveals heterogeneity of de novo fatty acid synthesis in cancer.

Metabolism has emerged as a key factor in homeostasis and disease including cancer. Yet, little is known about the heterogeneity of metabolic activity of cancer cells due to the lack of tools to directly probe it. Here, we present a novel method, 13C-SpaceM for spatial single-cell isotope tracing of glucose-dependent de novo lipogenesis. The method combines imaging mass spectrometry for spatially-resolved detection of 13C6-glucose-derived 13C label incorporated into esterified fatty acids with microscopy and computational methods for data integration and analysis. We validated 13C-SpaceM on a spatially-heterogeneous normoxia-hypoxia model of liver cancer cells. Investigating cultured cells, we revealed single-cell heterogeneity of lipogenic acetyl-CoA pool labelling degree upon ACLY knockdown that is hidden in the bulk analysis and its effect on synthesis of individual fatty acids. Next, we adapted 13C-SpaceM to analyze tissue sections of mice harboring isocitrate dehydrogenase (IDH)-mutant gliomas. We found a strong induction of de novo fatty acid synthesis in the tumor tissue compared to the surrounding brain. Comparison of fatty acid isotopologue patterns revealed elevated uptake of mono-unsaturated and essential fatty acids in the tumor. Furthermore, our analysis uncovered substantial spatial heterogeneity in the labelling of the lipogenic acetyl-CoA pool indicative of metabolic reprogramming during microenvironmental adaptation. Overall, 13C-SpaceM enables novel ways for spatial probing of metabolic activity at the single cell level. Additionally, this methodology provides unprecedented insight into fatty acid uptake, synthesis and modification in normal and cancerous tissues.

Targeting MYC induces lipid droplet accumulation by upregulation of HILPDA in clear cell renal cell carcinoma.

Metabolic reprogramming is critical during clear cell renal cell carcinoma (ccRCC) tumorigenesis, manifested by accumulation of lipid droplets (LDs), organelles that have emerged as new hallmarks of cancer. Yet, regulation of their biogenesis is still poorly understood. Here, we demonstrate that MYC inhibition in ccRCC cells lacking the von Hippel Lindau (VHL) gene leads to increased triglyceride content potentiating LD formation in a glutamine-dependent manner. Importantly, the concurrent inhibition of MYC signaling and glutamine metabolism prevented LD accumulation and reduced tumor burden in vivo. Furthermore, we identified the hypoxia-inducible lipid droplet-associated protein (HILPDA) as the key driver for induction of MYC-driven LD accumulation and demonstrated that conversely, proliferation, LD formation, and tumor growth are impaired upon its downregulation. Finally, analysis of ccRCC tissue as well as healthy renal control samples postulated HILPDA as a specific ccRCC biomarker. Together, these results provide an attractive approach for development of alternative therapeutic interventions for the treatment of this type of renal cancer.

Glycolytic flux-signaling controls mouse embryo mesoderm development.

How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here, we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic PFKFB3 (cytoPFKFB3). Overexpression of cytoPFKFB3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPFKFB3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.

The glycolytic enzyme ALDOA and the exon junction complex protein RBM8A are regulators of ribosomal biogenesis.

Cellular growth is a fundamental process of life and must be precisely controlled in multicellular organisms. Growth is crucially controlled by the number of functional ribosomes available in cells. The production of new ribosomes depends critically on the activity of RNA polymerase (RNAP) II in addition to the activity of RNAP I and III, which produce ribosomal RNAs. Indeed, the expression of both, ribosomal proteins and proteins required for ribosome assembly (ribosomal biogenesis factors), is considered rate-limiting for ribosome synthesis. Here, we used genetic screening to identify novel transcriptional regulators of cell growth genes by fusing promoters from a ribosomal protein gene (Rpl18) and from a ribosomal biogenesis factor (Fbl) with fluorescent protein genes (RFP, GFP) as reporters. Subsequently, both reporters were stably integrated into immortalized mouse fibroblasts, which were then transduced with a genome-wide sgRNA-CRISPR knockout library. Subsequently, cells with altered reporter activity were isolated by FACS and the causative sgRNAs were identified. Interestingly, we identified two novel regulators of growth genes. Firstly, the exon junction complex protein RBM8A controls transcript levels of the intronless reporters used here. By acute depletion of RBM8A protein using the auxin degron system combined with the genome-wide analysis of nascent transcription, we showed that RBM8A is an important global regulator of ribosomal protein transcripts. Secondly, we unexpectedly observed that the glycolytic enzyme aldolase A (ALDOA) regulates the expression of ribosomal biogenesis factors. Consistent with published observations that a fraction of this protein is located in the nucleus, this may be a mechanism linking transcription of growth genes to metabolic processes and possibly to metabolite availability.

Inhibition of fatty acid synthesis induces differentiation and reduces tumor burden in childhood neuroblastoma.

Many metabolic pathways, including lipid metabolism, are rewired in tumors to support energy and biomass production and to allow adaptation to stressful environments. Neuroblastoma is the second deadliest solid tumor in children. Genetic aberrations, as the amplification of the MYCN-oncogene, correlate strongly with disease progression. Yet, there are only a few molecular targets successfully exploited in the clinic. Here we show that inhibition of fatty acid synthesis led to increased neural differentiation and reduced tumor burden in neuroblastoma xenograft experiments independently of MYCN-status. This was accompanied by reduced levels of the MYCN or c-MYC oncoproteins and activation of ERK signaling. Importantly, the expression levels of genes involved in de novo fatty acid synthesis showed prognostic value for neuroblastoma patients. Our findings demonstrate that inhibition of de novo fatty acid synthesis is a promising pharmacological intervention strategy for the treatment of neuroblastoma independently of MYCN-status.

LXRα activation and Raf inhibition trigger lethal lipotoxicity in liver cancer.

The success of molecular therapies targeting specific metabolic pathways in cancer is often limited by the plasticity and adaptability of metabolic networks. Here we show that pharmacologically induced lipotoxicity represents a promising therapeutic strategy for the treatment of hepatocellular carcinoma (HCC). LXRα-induced liponeogenesis and Raf-1 inhibition are synthetic lethal in HCC owing to a toxic accumulation of saturated fatty acids. Raf-1 was found to bind and activate SCD1, and conformation-changing DFG-out Raf inhibitors could disrupt this interaction, thereby blocking fatty acid desaturation and inducing lethal lipotoxicity. Studies in genetically engineered and nonalcoholic steatohepatitis-induced HCC mouse models and xenograft models of human HCC revealed that therapies comprising LXR agonists and Raf inhibitors were well tolerated and capable of overcoming therapy resistance in HCC. Conceptually, our study suggests pharmacologically induced lipotoxicity as a new mode for metabolic targeting of liver cancer.

Greasing the Wheels of the Cancer Machine: The Role of Lipid Metabolism in Cancer.

Altered lipid metabolism is among the most prominent metabolic alterations in cancer. Enhanced synthesis or uptake of lipids contributes to rapid cancer cell growth and tumor formation. Lipids are a highly complex group of biomolecules that not only constitute the structural basis of biological membranes but also function as signaling molecules and an energy source. Here, we summarize recent evidence implicating altered lipid metabolism in different aspects of the cancer phenotype and discuss potential strategies by which targeting lipid metabolism could provide a therapeutic window for cancer treatment.

Non-canonical functions of enzymes facilitate cross-talk between cell metabolic and regulatory pathways.

The metabolic rewiring that occurs during cell transformation is a hallmark of cancer. It is diverse in different cancers as it reflects different combinations of oncogenic drivers, tumor suppressors, and the microenvironment. Metabolic rewiring is essential to cancer as it enables uncontrolled proliferation and adaptation to the fluctuating availability of nutrients and oxygen caused by poor access to the vasculature due to tumor growth and a foreign microenvironment encountered during metastasis. Increasing evidence now indicates that the metabolic state in cancer cells also plays a causal role in tumor growth and metastasis, for example through the action of oncometabolites, which modulate cell signaling and epigenetic pathways to promote malignancy. In addition to altering the metabolic state in cancer cells, some multifunctional enzymes possess non-metabolic functions that also contribute to cell transformation. Some multifunctional enzymes that are highly expressed in cancer, such as pyruvate kinase M2 (PKM2), have non-canonical functions that are co-opted by oncogenic signaling to drive proliferation and inhibit apoptosis. Other multifunctional enzymes that are frequently downregulated in cancer, such as fructose-bisphosphatase 1 (FBP1), are tumor suppressors, directly opposing mitogenic signaling via their non-canonical functions. In some cases, the enzymatic and non-canonical roles of these enzymes are functionally linked, making the modulation of non-metabolic cellular processes dependent on the metabolic state of the cell.

Spatiotemporal Analysis of a Glycolytic Activity Gradient Linked to Mouse Embryo Mesoderm Development.

How metabolism is rewired during embryonic development is still largely unknown, as it remains a major technical challenge to resolve metabolic activities or metabolite levels with spatiotemporal resolution. Here, we investigated metabolic changes during development of organogenesis-stage mouse embryos, focusing on the presomitic mesoderm (PSM). We measured glycolytic labeling kinetics from 13C-glucose tracing experiments and detected elevated glycolysis in the posterior, more undifferentiated PSM. We found evidence that the spatial metabolic differences are functionally relevant during PSM development. To enable real-time quantification of a glycolytic metabolite with spatiotemporal resolution, we generated a pyruvate FRET-sensor reporter mouse line. We revealed dynamic changes in cytosolic pyruvate levels as cells transit toward a more anterior PSM state. Combined, our approach identifies a gradient of glycolytic activity across the PSM, and we provide evidence that these spatiotemporal metabolic changes are intrinsically linked to PSM development and differentiation.

Differential water permeability and regulation of three aquaporin 4 isoforms.

Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the brain (M1, M23, Mz). The M23 isoform gave rise to square arrays when expressed in Xenopus laevis oocytes. The relative unit water permeability differed significantly between the isoforms in the order of M1 > Mz > M23. None of the three isoforms were permeable to small osmolytes nor were they affected by changes in external K(+) concentration. Upon protein kinase C (PKC) activation, oocytes expressing the three isoforms demonstrated rapid reduction of water permeability, which correlated with AQP4 internalization. The M23 isoform was more sensitive to PKC regulation than the longer isoforms and was internalized significantly faster. Our results suggest a specific role for square array formation.