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.

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.

A multidimensional atlas of human glioblastoma-like organoids reveals highly coordinated molecular networks and effective drugs.

Recent advances in the genomics of glioblastoma (GBM) led to the introduction of molecular neuropathology but failed to translate into treatment improvement. This is largely attributed to the genetic and phenotypic heterogeneity of GBM, which are considered the major obstacle to GBM therapy. Here, we use advanced human GBM-like organoid (LEGO: Laboratory Engineered Glioblastoma-like Organoid) models and provide an unprecedented comprehensive characterization of LEGO models using single-cell transcriptome, DNA methylome, metabolome, lipidome, proteome, and phospho-proteome analysis. We discovered that genetic heterogeneity dictates functional heterogeneity across molecular layers and demonstrates that NF1 mutation drives mesenchymal signature. Most importantly, we found that glycerol lipid reprogramming is a hallmark of GBM, and several targets and drugs were discovered along this line. We also provide a genotype-based drug reference map using LEGO-based drug screen. This study provides new human GBM models and a research path toward effective GBM therapy.

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.

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.

Formate promotes invasion and metastasis in reliance on lipid metabolism.

Metabolic rewiring is essential for cancer onset and progression. We previously showed that one-carbon metabolism-dependent formate production often exceeds the anabolic demand of cancer cells, resulting in formate overflow. Furthermore, we showed that increased extracellular formate concentrations promote the in vitro invasiveness of glioblastoma cells. Here, we substantiate these initial observations with ex vivo and in vivo experiments. We also show that exposure to exogeneous formate can prime cancer cells toward a pro-invasive phenotype leading to increased metastasis formation in vivo. Our results suggest that the increased local formate concentration within the tumor microenvironment can be one factor to promote metastases. Additionally, we describe a mechanistic interplay between formate-dependent increased invasiveness and adaptations of lipid metabolism and matrix metalloproteinase activity. Our findings consolidate the role of formate as pro-invasive metabolite and warrant further research to better understand the interplay between formate and lipid metabolism.

LRP8-mediated selenocysteine uptake is a targetable vulnerability in MYCN-amplified neuroblastoma.

Ferroptosis has emerged as an attractive strategy in cancer therapy. Understanding the operational networks regulating ferroptosis may unravel vulnerabilities that could be harnessed for therapeutic benefit. Using CRISPR-activation screens in ferroptosis hypersensitive cells, we identify the selenoprotein P (SELENOP) receptor, LRP8, as a key determinant protecting MYCN-amplified neuroblastoma cells from ferroptosis. Genetic deletion of LRP8 leads to ferroptosis as a result of an insufficient supply of selenocysteine, which is required for the translation of the antiferroptotic selenoprotein GPX4. This dependency is caused by low expression of alternative selenium uptake pathways such as system Xc- . The identification of LRP8 as a specific vulnerability of MYCN-amplified neuroblastoma cells was confirmed in constitutive and inducible LRP8 knockout orthotopic xenografts. These findings disclose a yet-unaccounted mechanism of selective ferroptosis induction that might be explored as a therapeutic strategy for high-risk neuroblastoma and potentially other MYCN-amplified entities.

USP28 controls SREBP2 and the mevalonate pathway to drive tumour growth in squamous cancer.

SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.

Cholesterol atlas of tumor microenvironment provides route to improved CAR-T therapy.

Cholesterol is essential for the ability of cytotoxic T cells to eliminate cancer cells. In this issue of Cancer Cell, Yan et al. reveal how intra-tumoral cholesterol deficiency contributes to T cell exhaustion by inhibiting mTORC1 signaling. Moreover, they demonstrate that increasing cholesterol levels in chimeric antigen receptor (CAR)-T cells by blocking liver X receptor (LXR) leads to improved anti-tumor function.

Palmitate paves the way to lung metastasis.

New findings by Altea-Manzano et al. demonstrate that primary breast tumours or a high-fat diet induce palmitate secretion by alveolar type 2 (AT2) cells to prepare a premetastatic niche that promotes lung metastasis. Palmitate is utilised by cancer cells via carnitine palmitoyltransferase 1a (CPT1a) and lysine acetyltransferase 2a (KAT2a) to acetylate nuclear factor-κB (NF-κB)/p65. Blocking these enzymes reduced metastasis formation in both lean and high-fat diet mice.

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.

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.

c-Myc plays a key role in IFN-γ-induced persistence of Chlamydia trachomatis.

Chlamydia trachomatis (Ctr) can persist over extended times within their host cell and thereby establish chronic infections. One of the major inducers of chlamydial persistence is interferon-gamma (IFN-γ) released by immune cells as a mechanism of immune defence. IFN-γ activates the catabolic depletion of L-tryptophan (Trp) via indoleamine-2,3-dioxygenase (IDO), resulting in persistent Ctr. Here, we show that IFN-γ induces the downregulation of c-Myc, the key regulator of host cell metabolism, in a STAT1-dependent manner. Expression of c-Myc rescued Ctr from IFN-γ-induced persistence in cell lines and human fallopian tube organoids. Trp concentrations control c-Myc levels most likely via the PI3K-GSK3ß axis. Unbiased metabolic analysis revealed that Ctr infection reprograms the host cell tricarboxylic acid (TCA) cycle to support pyrimidine biosynthesis. Addition of TCA cycle intermediates or pyrimidine/purine nucleosides to infected cells rescued Ctr from IFN-γ-induced persistence. Thus, our results challenge the longstanding hypothesis of Trp depletion through IDO as the major mechanism of IFN-γ-induced metabolic immune defence and significantly extends the understanding of the role of IFN-γ as a broad modulator of host cell metabolism.

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.

Acute systemic knockdown of Atg7 is lethal and causes pancreatic destruction in shRNA transgenic mice.

The notion that macroautophagy/autophagy is a potentially attractive therapeutic target for a variety of diseases, including cancer, largely stems from pre-clinical mouse studies. Most of these examine the effects of irreversible and organ confined autophagy deletion using site specific Cre-loxP recombination of the essential autophagy regulating genes Atg7 or Atg5. Model systems with the ability to impair autophagy systemically and reversibly at all disease stages would allow a more realistic approach to evaluate the consequences of authophagy inhibition as a therapeutic concept and its potential side effects. Here, we present shRNA transgenic mice that via doxycycline (DOX) regulable expression of a highly efficient miR30-E-based shRNA enabled knockdown of Atg7 simultaneously in the majority of organs, with the brain and spleen being noteable exceptions. Induced animals deteriorated rapidly and experienced profound destruction of the exocrine pancreas, severe hypoglycemia and depletion of hepatic glycogen storages. Cessation of DOX application restored apparent health, glucose homeostasis and pancreatic integrity. In a similar Atg5 knockdown model we neither observed loss of pancreatic integrity nor diminished survival after DOX treatment, but identified histological changes consistent with steatohepatitis and hepatic fibrosis in the recovery period after termination of DOX. Regulable Atg7-shRNA mice are valuable tools that will enable further studies on the role of autophagy impairment at various disease stages and thereby help to evaluate the consequences of acute autophagy inhibition as a therapeutic concept.Abbreviations: ACTB: actin, beta; AMY: amylase complex; ATG4B: autophagy related 4B, cysteine peptidase; ATG5: autophagy related 5; ATG7: autophagy related 7; Cag: CMV early enhancer/chicken ACTB promoter; Col1a1: collagen, type I, alpha 1; Cre: cre recombinase; DOX: doxycycline; GCG: glucagon; GFP: green fluorescent protein; INS: insulin; LC3: microtubule-associated protein 1 light chain 3; miR30-E: optimized microRNA backbone; NAFLD: non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis; PNLIP: pancreatic lipase; rtTA: reverse tetracycline transactivator protein; SQSTM1/p62: sequestome 1; TRE: tetracycline responsive element.

Integrated Metabolomics and Transcriptomics Analysis of Monolayer and Neurospheres from Established Glioblastoma Cell Lines.

Altered metabolic processes contribute to carcinogenesis by modulating proliferation, survival and differentiation. Tumours are composed of different cell populations, with cancer stem-like cells being one of the most prominent examples. This specific pool of cells is thought to be responsible for cancer growth and recurrence and plays a particularly relevant role in glioblastoma (GBM), the most lethal form of primary brain tumours. Here, we have analysed the transcriptome and metabolome of an established GBM cell line (U87) and a patient-derived GBM stem-like cell line (NCH644) exposed to neurosphere or monolayer culture conditions. By integrating transcriptome and metabolome data, we identified key metabolic pathways and gene signatures that are associated with stem-like and differentiated states in GBM cells, and demonstrated that neurospheres and monolayer cells differ substantially in their metabolism and gene regulation. Furthermore, arginine biosynthesis was identified as the most significantly regulated pathway in neurospheres, although individual nodes of this pathway were distinctly regulated in the two cellular systems. Neurosphere conditions, as opposed to monolayer conditions, cause a transcriptomic and metabolic rewiring that may be crucial for the regulation of stem-like features, where arginine biosynthesis may be a key metabolic pathway. Additionally, TCGA data from GBM patients showed significant regulation of specific components of the arginine biosynthesis pathway, providing further evidence for the importance of this metabolic pathway in GBM.

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.

Implementation of CRISPR/Cas9 Genome Editing to Generate Murine Lung Cancer Models That Depict the Mutational Landscape of Human Disease.

Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53 fl/fl :lsl-KRas G12D/wt . Developing tumors were indistinguishable from Trp53 fl/fl :lsl-KRas G12D/ wt -derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research.