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.

p53 deficient breast cancer cells reprogram preadipocytes toward tumor-protective immunomodulatory cells.

The TP53 gene is mutated in approximately 30% of all breast cancer cases. Adipocytes and preadipocytes, which constitute a substantial fraction of the stroma of normal mammary tissue and breast tumors, undergo transcriptional, metabolic, and phenotypic reprogramming during breast cancer development and play an important role in tumor progression. We report here that p53 loss in breast cancer cells facilitates the reprogramming of preadipocytes, inducing them to acquire a unique transcriptional and metabolic program that combines impaired adipocytic differentiation with augmented cytokine expression. This, in turn, promotes the establishment of an inflammatory tumor microenvironment, including increased abundance of Ly6C+ and Ly6G+ myeloid cells and elevated expression of the immune checkpoint ligand PD-L1. We also describe a potential gain-of-function effect of common p53 missense mutations on the inflammatory reprogramming of preadipocytes. Altogether, our study implicates p53 deregulation in breast cancer cells as a driver of tumor-supportive adipose tissue reprogramming, expanding the network of non-cell autonomous mechanisms whereby p53 dysfunction may promote cancer. Further elucidation of the interplay between p53 and adipocytes within the tumor microenvironment may suggest effective therapeutic targets for the treatment of breast cancer patients.

Hydropersulfides inhibit lipid peroxidation and ferroptosis by scavenging radicals.

Ferroptosis is a type of cell death caused by radical-driven lipid peroxidation, leading to membrane damage and rupture. Here we show that enzymatically produced sulfane sulfur (S0) species, specifically hydropersulfides, scavenge endogenously generated free radicals and, thereby, suppress lipid peroxidation and ferroptosis. By providing sulfur for S0 biosynthesis, cysteine can support ferroptosis resistance independently of the canonical GPX4 pathway. Our results further suggest that hydropersulfides terminate radical chain reactions through the formation and self-recombination of perthiyl radicals. The autocatalytic regeneration of hydropersulfides may explain why low micromolar concentrations of persulfides suffice to produce potent cytoprotective effects on a background of millimolar concentrations of glutathione. We propose that increased S0 biosynthesis is an adaptive cellular response to radical-driven lipid peroxidation, potentially representing a primordial radical protection system.

FSP1 is a glutathione-independent ferroptosis suppressor.

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been thought to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is crucial to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10, CoQ10): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1-CoQ10-NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis.

Metformin Regulates the miR-205/VEGFA Axis in Renal Cell Carcinoma Cells: Exploring a Clinical Synergism with Tyrosine Kinase Inhibitors.

INTRODUCTION: Metformin (MF) intake could be associated with a favorable outcome in sunitinib (SUT)- and axitinib (AX)-treated clear cell renal cell carcinoma (ccRCC) patients. Functionally, MF induces miR-205, a microRNA serving as a tumor suppressor in several cancers. METHODS: Real-time quantitative PCR, viability assays, and Western blotting analyzed MF and SUT/AX effects in RCC4 and 786-O cells. A tetracycline-inducible overexpression model was used to study the role of miR-205 and its known target gene, VEGFA. We analyzed miR-205 and VEGFA within a public and an in-house ccRCC cohort. Human umbilical vein endothelial cell (HUVEC) sprouting assays examined miR-205 effects on angiogenesis initiation. To determine the influence of the von Hippel-Lindau tumor suppressor (VHL), we examined VHLwt reexpressing RCC4 and 786-O cells. RESULTS: Viability assays confirmed a sensitizing effect of MF toward SUT/AX in RCC4 and 786-O cells. Overexpression of miR-205 diminished VEGFA expression - as did treatment with MF. Tumor tissue displayed a downregulation of miR-205 and an upregulation of VEGFA. Accordingly, miR-205 caused less and shorter vessel sprouts in HUVEC assays. Finally, VHLwt-expressing RCC4 and 786-O cells displayed higher miR-205 and lower VEGFA levels. CONCLUSION: Our results support the protective role of MF in ccRCC and offer functional insights into the clinical synergism with tyrosine kinase inhibitors.

Protein kinase D1 deletion in adipocytes enhances energy dissipation and protects against adiposity.

Nutrient overload in combination with decreased energy dissipation promotes obesity and diabetes. Obesity results in a hormonal imbalance, which among others activates G protein-coupled receptors utilizing diacylglycerol (DAG) as secondary messenger. Protein kinase D1 (PKD1) is a DAG effector, which integrates multiple nutritional and hormonal inputs, but its physiological role in adipocytes is unknown. Here, we show that PKD1 promotes lipogenesis and suppresses mitochondrial fragmentation, biogenesis, respiration, and energy dissipation in an AMP-activated protein kinase (AMPK)-dependent manner. Moreover, mice lacking PKD1 in adipocytes are resistant to diet-induced obesity due to elevated energy expenditure. Beiging of adipocytes promotes energy expenditure and counteracts obesity. Consistently, deletion of PKD1 promotes expression of the ß3-adrenergic receptor (ADRB3) in a CCAAT/enhancer binding protein (C/EBP)-α- and δ-dependent manner, which leads to the elevated expression of beige markers in adipocytes and subcutaneous adipose tissue. Finally, deletion of PKD1 in adipocytes improves insulin sensitivity and ameliorates liver steatosis. Thus, depletion of PKD1 in adipocytes increases energy dissipation by several complementary mechanisms and might represent an attractive strategy to treat obesity and its related complications.

SOAT1: A Suitable Target for Therapy in High-Grade Astrocytic Glioma?

Targeting molecular alterations as an effective treatment for isocitrate dehydrogenase-wildtype glioblastoma (GBM) patients has not yet been established. Sterol-O-Acyl Transferase 1 (SOAT1), a key enzyme in the conversion of endoplasmic reticulum cholesterol to esters for storage in lipid droplets (LD), serves as a target for the orphan drug mitotane to treat adrenocortical carcinoma. Inhibition of SOAT1 also suppresses GBM growth. Here, we refined SOAT1-expression in GBM and IDH-mutant astrocytoma, CNS WHO grade 4 (HGA), and assessed the distribution of LD in these tumors. Twenty-seven GBM and three HGA specimens were evaluated by multiple GFAP, Iba1, IDH1 R132H, and SOAT1 immunofluorescence labeling as well as Oil Red O staining. To a small extent SOAT1 was expressed by tumor cells in both tumor entities. In contrast, strong expression was observed in glioma-associated macrophages. Triple immunofluorescence labeling revealed, for the first time, evidence for SOAT1 colocalization with Iba1 and IDH1 R132H, respectively. Furthermore, a notable difference in the amount of LD between GBM and HGA was observed. Therefore, SOAT1 suppression might be a therapeutic option to target GBM and HGA growth and invasiveness. In addition, the high expression in cells related to neuroinflammation could be beneficial for a concomitant suppression of protumoral microglia/macrophages.

Ferroptosis: The Greasy Side of Cell Death.

Ferroptosis is a form of cell death that requires phospholipid peroxidation and has attracted increased attention, both as a means to eradicate tumors resistant to standard chemotherapy and for its potential contribution to tissue damage such as in ischemia/reperfusion. The center stage taken by phospholipid peroxidation in ferroptosis is highlighted by recent discoveries that demonstrate an intricate regulation of both the metabolism of polyunsaturated fatty acids as well as mechanisms leading to their oxidation. These metabolic steps converge at the point of ferroptosis execution through mechanisms that are now only starting to be understood. In this short review, we provide an appraisal of some of the recent advances in the understanding of the ferroptosis process and also provide some perspectives of where this knowledge could take us.

Connecting lysosomes and mitochondria - a novel role for lipid metabolism in cancer cell death.

BACKGROUND: The understanding of lysosomes has been expanded in recent research way beyond their view as cellular trash can. Lysosomes are pivotal in regulating metabolism, endocytosis and autophagy and are implicated in cancer. Recently it was discovered that the lysosomal V-ATPase, which is known to induce apoptosis, interferes with lipid metabolism in cancer, yet the interplay between these organelles is poorly understood. METHODS: LC-MS/MS analysis was performed to investigate lipid distribution in cells. Cell survival and signaling pathways were analyzed by means of cell biological methods (qPCR, Western Blot, flow cytometry, CellTiter-Blue). Mitochondrial structure was analyzed by confocal imaging and electron microscopy, their function was determined by flow cytometry and seahorse measurements. RESULTS: Our data reveal that interfering with lysosomal function changes composition and subcellular localization of triacylglycerids accompanied by an upregulation of PGC1α and PPARα expression, master regulators of energy and lipid metabolism. Furthermore, cardiolipin content is reduced driving mitochondria into fission, accompanied by a loss of membrane potential and reduction in oxidative capacity, which leads to a deregulation in cellular ROS and induction of mitochondria-driven apoptosis. Additionally, cells undergo a metabolic shift to glutamine dependency, correlated with the fission phenotype and sensitivity to lysosomal inhibition, most prominent in Ras mutated cells. CONCLUSION: This study sheds mechanistic light on a largely uninvestigated triangle between lysosomes, lipid metabolism and mitochondrial function. Insight into this organelle crosstalk increases our understanding of mitochondria-driven cell death. Our findings furthermore provide a first hint on a connection of Ras pathway mutations and sensitivity towards lysosomal inhibitors.

Flicking the Warburg switch-tyrosine phosphorylation of pyruvate dehydrogenase kinase regulates mitochondrial activity in cancer cells.

In this issue of Molecular Cell, Hitosugi et al. (2011) show that the switch from oxidative phosphorylation to glycolysis in cancer cells is regulated by tyrosine phosphorylation of PDHK1.

How cancer metabolism is tuned for proliferation and vulnerable to disruption.

Cancer metabolism has received a substantial amount of interest over the past decade. The advances in analytical tools have, along with the rapid progress of cancer genomics, generated an increasingly complex understanding of metabolic reprogramming in cancer. As numerous connections between oncogenic signalling pathways and metabolic activities emerge, the importance of metabolic reprogramming in cancer is being increasingly recognized. The identification of metabolic weaknesses of cancer cells has been used to create strategies for treating cancer, but there are still challenges to be faced in bringing the drugs that target cancer metabolism to the clinic.

Metabotypes of breast cancer cell lines revealed by non-targeted metabolomics.

We present an analysis of intracellular metabolism by non-targeted, high-throughput metabolomics profiling of 18 breast cell lines. We profiled >900 putatively annotated metabolite ions for >100 samples collected under both normoxic and hypoxic conditions and revealed extensive heterogeneity across all metabolic pathways and cell lines. Cell line-specific metabolome profiles dominated over patterns associated with malignancy or with the clinical nomenclature of breast cancer cells. Such characteristic metabolome profiles were reproducible across different laboratories and experiments and exhibited mild to robust changes with change in experimental conditions. To extract a functional overview of cell line heterogeneity, we devised an unsupervised metabotyping procedure that for each pathway automatically recognized metabolic types from metabolome data and assigned cell lines. Our procedure provided a condensed yet global representation of cell line metabolism, revealing the fine structure of metabolic heterogeneity across all tested pathways and cell lines. In follow-up experiments on selected pathways, we confirmed that different metabolic types correlated to differences in the underlying fluxes and difference sensitivity to gene knockdown or pharmacological inhibition. Thus, the identified metabotypes recapitulated functional differences at the pathway level. Metabotyping provides a powerful compression of multi-dimensional data that preserves functional information and serves as a resource for reconciling or understanding heterogeneous metabolic phenotypes or response to inhibition of metabolic pathways.

The Role of Glucose and Lipid Metabolism in Growth and Survival of Cancer Cells.

One of the prerequisites for cell growth and proliferation is the synthesis of macromolecules, including proteins, nucleic acids and lipids. Cells have to alter their metabolism to allow the production of metabolic intermediates that are the precursors for biomass production. It is now evident that oncogenic signalling pathways target metabolic processes on several levels and metabolic reprogramming has emerged as a hallmark of cancer. The increased metabolic demand of cancer cells also produces selective dependencies that could be targeted for therapeutic intervention. Understanding the role of glucose and lipid metabolism in supporting cancer cell growth and survival is crucial to identify essential processes that could provide therapeutic windows for cancer therapy.

Functional screening identifies MCT4 as a key regulator of breast cancer cell metabolism and survival.

Metabolic reprogramming in cancer enhances macromolecule biosynthesis and supports cell survival. Oncogenic drivers affect metabolism by altering distinct metabolic processes and render cancer cells sensitive to perturbations of the metabolic network. This study aimed to identify selective metabolic dependencies in breast cancer by investigating 17 breast cancer cells lines representative of the genetic diversity of the disease. Using a functional screen, we demonstrate here that monocarboxylate transporter 4 (MCT4) is an important regulator of breast cancer cell survival. MCT4 supports pH maintenance, lactate secretion and non-oxidative glucose metabolism in breast cancer cells. Moreover, MCT4 depletion caused an increased dependence of cancer cells on mitochondrial respiration and glutamine metabolism. MCT4 depletion reduced the ability of breast cancer cells to grow in a three-dimensional (3D) matrix or as multilayered spheroids. Moreover, MCT4 expression is regulated by the PI3K-Akt signalling pathway and highly expressed in HER2-positive breast cancers. These results suggest that MCT4 is a potential therapeutic target in defined breast cancer subtypes and reveal novel avenues for combination treatment.

A computational study of the Warburg effect identifies metabolic targets inhibiting cancer migration.

Over the last decade, the field of cancer metabolism has mainly focused on studying the role of tumorigenic metabolic rewiring in supporting cancer proliferation. Here, we perform the first genome-scale computational study of the metabolic underpinnings of cancer migration. We build genome-scale metabolic models of the NCI-60 cell lines that capture the Warburg effect (aerobic glycolysis) typically occurring in cancer cells. The extent of the Warburg effect in each of these cell line models is quantified by the ratio of glycolytic to oxidative ATP flux (AFR), which is found to be highly positively associated with cancer cell migration. We hence predicted that targeting genes that mitigate the Warburg effect by reducing the AFR may specifically inhibit cancer migration. By testing the anti-migratory effects of silencing such 17 top predicted genes in four breast and lung cancer cell lines, we find that up to 13 of these novel predictions significantly attenuate cell migration either in all or one cell line only, while having almost no effect on cell proliferation. Furthermore, in accordance with the predictions, a significant reduction is observed in the ratio between experimentally measured ECAR and OCR levels following these perturbations. Inhibiting anti-migratory targets is a promising future avenue in treating cancer since it may decrease cytotoxic-related side effects that plague current anti-proliferative treatments. Furthermore, it may reduce cytotoxic-related clonal selection of more aggressive cancer cells and the likelihood of emerging resistance.

Genome-wide analysis of FOXO3 mediated transcription regulation through RNA polymerase II profiling.

Forkhead box O (FOXO) transcription factors are key players in diverse cellular processes affecting tumorigenesis, stem cell maintenance and lifespan. To gain insight into the mechanisms of FOXO-regulated target gene expression, we studied genome-wide effects of FOXO3 activation. Profiling RNA polymerase II changes shows that FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNA polymerase II ChIP-seq profiles demonstrates FOXO3 to act as a transcriptional activator. Furthermore, this analysis reveals a significant part of FOXO3 gene regulation proceeds through enhancer regions. FOXO3 binds to pre-existing enhancers and further activates these enhancers as shown by changes in histone acetylation and RNA polymerase II recruitment. In addition, FOXO3-mediated enhancer activation correlates with regulation of adjacent genes and pre-existence of chromatin loops between FOXO3 bound enhancers and target genes. Combined, our data elucidate how FOXOs regulate gene transcription and provide insight into mechanisms by which FOXOs can induce different gene expression programs depending on chromatin architecture.

Lipids as mediators of cancer progression and metastasis.

Metastasis formation is a complex process, involving multiple crucial steps, which are controlled by different regulatory mechanisms. In this context, the contribution of cancer metabolism to the metastatic cascade is being increasingly recognized. This Review focuses on changes in lipid metabolism that contribute to metastasis formation in solid tumors. We discuss the molecular mechanisms by which lipids induce a pro-metastatic phenotype and explore the role of lipids in response to oxidative stress and as signaling molecules. Finally, we reflect on potential avenues to target lipid metabolism to improve the treatment of metastatic cancers.

Interactions of Fatty Acid and Cholesterol Metabolism with Cellular Stress Response Pathways in Cancer.

Lipids have essential functions as structural components of cellular membranes, as efficient energy storage molecules, and as precursors of signaling mediators. While deregulated glucose and amino acid metabolism in cancer have received substantial attention, the roles of lipids in the metabolic reprogramming of cancer cells are less well understood. However, since the first description of de novo fatty acid biosynthesis in cancer tissues almost 70 years ago, numerous studies have investigated the complex functions of altered lipid metabolism in cancer. Here, we will summarize the mechanisms by which oncogenic signaling pathways regulate fatty acid and cholesterol metabolism to drive rapid proliferation and protect cancer cells from environmental stress. The review also discusses the role of fatty acid metabolism in metabolic plasticity required for the adaptation to changing microenvironments during cancer progression and the connections between fatty acid and cholesterol metabolism and ferroptosis.

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.