CORDIS - EU research results
CORDIS

Cancer Cellular Metabolism across Space and Time

Periodic Reporting for period 4 - CancerFluxome (Cancer Cellular Metabolism across Space and Time)

Reporting period: 2021-08-01 to 2023-01-31

Cancer cells develop remarkably distinct metabolism compared to normal cells. Evidence for alterations in metabolic activity in malignant cells goes back almost a century ago to the discovery of the ‘Warburg effect’. The recent resurgence of interest in the field of cancer metabolism involved numerous findings of metabolic alterations in a variety of pathways in cancer cells, evidence for oncogenes and tumour suppressors regulating metabolic enzymes and specific oncogenic mutations in metabolic genes. While extensively studied, our current view of cancer cellular metabolism is fundamentally limited by lack of information on variability in metabolic activity between distinct subcellular compartments and cells. Specifically, while mitochondrial bioenergetics and biosynthesis play a major role in tumorigenesis, we typically lack basic understanding of the metabolic interplay between cytoplasm is and mitochondria; and how cells adapt central metabolic fluxes to fulfil the changing energetic and anabolic demands throughout the cell-cycle. Analysing the rate of metabolic reactions (i.e. metabolic flux) via isotope tracing is a central technique in cancer metabolic research. However, applied on a population of cells that are heterogeneous in terms of cell-cycle phase and not accounting for subcellular compartments confounds flux estimations and provides an overly simplified view of cellular metabolism

The CancerFluxome project focused on the development a spatio-temporal approach for quantifying metabolic activities in distinct subcellular compartments as well as their cell-cycle dynamics, combining a variety of experimental and computational techniques – including, mass-spectrometry based isotope tracing with cell synchronization, rapid cellular fractionation, and metabolic network modelling. The developed methods enable to infer metabolite concentrations and fluxes in mitochondrial versus cytosolic metabolic processes under physiological cellular conditions. Furthermore, the developed methods enable to explore flux dynamics as cells progress throughout the cell cycle, providing important information on metabolic activities that are masked by traditional measurement and modelling approaches. The developed methods were applied to study flux reprogramming in one-carbon metabolism in cancer cells - a metabolic systems that plays a major role in cancer cell proliferation and has long been a target for chemotherapy drugs. Our studies challenged the view of how folate is metabolized in cancer cells and specifically on the role of mitochondrial versus cytosolic metabolic activities. It further revealed enzymes in one-carbon metabolism as novel targets for cancer therapy.

From a basic science perspective, this project challenged our current view of cellular metabolism, zooming in from the level of average quantities over a cell-population to a high-resolution, spatio-temporal cellular view of metabolic activities. The developed methods for spatio-temporal metabolic analysis are expected to greatly contribute to the ever-growing research effort in cancer- and immune-cell metabolism. From a clinical front, our work revealed novel potential anti-cancer drug targets whose clinical potential will require further research.
We developed a temporal-fluxomics approach to derive a comprehensive and quantitative view of alterations in metabolic fluxes throughout the mammalian cell cycle (Ahn et al., Molecular Systems Biology, 2017). We developed a spatial-fluxomics approach to infer metabolic fluxes in mitochondria and cytosol, combining isotope tracing, rapid subcellular fractionation, LC-MS-based metabolomics, computational deconvolution, and metabolic network modeling (Lee et al., Nature Communications 2019.). Considering the technical complexity in performing rapid cell fractionation and optimizing the approach for specific cells of interest, we developed a complementary approach for inferring subcellular metabolic activities strictly based on measurements performed with intact cells under physiological conditions. A revision of our paper on this topic by Stern et al is under review in Nature Communications.

Performing temporal-fluxomics analysis of cultured HeLa cells shows, for the first time, that TCA cycle fluxes are rewired as cells progress through the cell cycle with complementary oscillations of glucose versus glutamine-derived fluxes. We applied our spatial-fluxomics method to investigate the interplay between mitochondrial, nuclear, and cytosolic reactions involved in the production of acetyl-CoA in cancer cell lines, an important metabolic precursor for energy production, fatty-acid biosynthesis, and protein acetylation.

We applied different variants of spatial-fluxomics analysis to analyze subcellular compartmentalized metabolic fluxes and their regulation in a folate acid metabolism across cancer cells. In one study, we revealed a novel dependence of cancer cells specifically on the cytosolic serine hydroxymethyltransferase (SHMT1) in cancer cells – in contrast to the acceptable view of the mitochondrial pathway serving as the as the major source of one-carbon units in cancer cells (Lee et al., Cell Metabolism 2021). In another study, we quantified flux through mitochondrial glycine -cleavage system in cancer cell lines (Mukha et al, Cell Metabolism 2022). We found substantial and previously uncharacterized high flux in hepatocellular carcinoma (HCC) cells, supporting nucleotide biosynthesis.
A most significant achievement of the ERC project was the development of methodologies for exploring cellular metabolic activities at a spatio-temporal resolution. Specifically, our methods enable to infer metabolite concentrations and fluxes in distinct subcellular fractions, focusing on mitochondrial versus cytosolic metabolic processes. These methods further enables to explore flux dynamics as cells progress throughout the cell cycle, providing important information on metabolic activities that are masked by traditional approaches. An additional achievement is the application of such methods to study flux alterations in one-carbon metabolism in cancer cells. These studies challenged our view of folate metabolism in cancer cells and the role of mitochondrial versus cytosolic metabolic activities. It further revealed enzymes in one-carbon metabolism as novel targets for cancer therapy.
erc-fig.jpg