ALDH1L2 regulation of formate, formyl-methionine, and ROS controls cancer cell migration and metastasis.

Mitochondrial 10-formyltetrahydrofolate (10-formyl-THF) is utilized by three mitochondrial enzymes to produce formate for nucleotide synthesis, NADPH for antioxidant defense, and formyl-methionine (fMet) to initiate mitochondrial mRNA translation. One of these enzymes-aldehyde dehydrogenase 1 family member 2 (ALDH1L2)-produces NADPH by catabolizing 10-formyl-THF into CO2 and THF. Using breast cancer cell lines, we show that reduction of ALDH1L2 expression increases ROS levels and the production of both formate and fMet. Both depletion of ALDH1L2 and direct exposure to formate result in enhanced cancer cell migration that is dependent on the expression of the formyl-peptide receptor (FPR). In various tumor models, increased ALDH1L2 expression lowers formate and fMet accumulation and limits metastatic capacity, while human breast cancer samples show a consistent reduction of ALDH1L2 expression in metastases. Together, our data suggest that loss of ALDH1L2 can support metastatic progression by promoting formate and fMet production, resulting in enhanced FPR-dependent signaling.

The dietary sweetener sucralose is a negative modulator of T cell-mediated responses.

Artificial sweeteners are used as calorie-free sugar substitutes in many food products and their consumption has increased substantially over the past years1. Although generally regarded as safe, some concerns have been raised about the long-term safety of the consumption of certain sweeteners2-5. In this study, we show that the intake of high doses of sucralose in mice results in immunomodulatory effects by limiting T cell proliferation and T cell differentiation. Mechanistically, sucralose affects the membrane order of T cells, accompanied by a reduced efficiency of T cell receptor signalling and intracellular calcium mobilization. Mice given sucralose show decreased CD8+ T cell antigen-specific responses in subcutaneous cancer models and bacterial infection models, and reduced T cell function in models of T cell-mediated autoimmunity. Overall, these findings suggest that a high intake of sucralose can dampen T cell-mediated responses, an effect that could be used in therapy to mitigate T cell-dependent autoimmune disorders.

PHGDH is required for germinal center formation and is a therapeutic target in MYC-driven lymphoma.

The synthesis of serine from glucose is a key metabolic pathway supporting cellular proliferation in healthy and malignant cells. Despite this, the role that this aspect of metabolism plays in germinal center biology and pathology is not known. Here, we performed a comprehensive characterization of the role of the serine synthesis pathway in germinal center B cells and lymphomas derived from these cells. We demonstrate that upregulation of a functional serine synthesis pathway is a metabolic hallmark of B cell activation and the germinal center reaction. Inhibition of phosphoglycerate dehydrogenase (PHGDH), the first and rate-limiting enzyme in this pathway, led to defective germinal formation and impaired high-affinity antibody production. In addition, overexpression of enzymes involved in serine synthesis was a characteristic of germinal center B cell-derived lymphomas, with high levels of expression being predictive of reduced overall survival in diffuse large B cell lymphoma. Inhibition of PHGDH induced apoptosis in lymphoma cells, reducing disease progression. These findings establish PHGDH as a critical player in humoral immunity and a clinically relevant target in lymphoma.

The role of ROS in tumour development and progression.

Eukaryotic cells have developed complex systems to regulate the production and response to reactive oxygen species (ROS). Different ROS control diverse aspects of cell behaviour from signalling to death, and deregulation of ROS production and ROS limitation pathways are common features of cancer cells. ROS also function to modulate the tumour environment, affecting the various stromal cells that provide metabolic support, a blood supply and immune responses to the tumour. Although it is clear that ROS play important roles during tumorigenesis, it has been difficult to reliably predict the effect of ROS modulating therapies. We now understand that the responses to ROS are highly complex and dependent on multiple factors, including the types, levels, localization and persistence of ROS, as well as the origin, environment and stage of the tumours themselves. This increasing understanding of the complexity of ROS in malignancies will be key to unlocking the potential of ROS-targeting therapies for cancer treatment.

The impact of physiological metabolite levels on serine uptake, synthesis and utilization in cancer cells.

Serine is a non-essential amino acid that is critical for tumour proliferation and depletion of circulating serine results in reduced tumour growth and increased survival in various cancer models. While many cancer cells cultured in a standard tissue culture medium depend on exogenous serine for optimal growth, here we report that these cells are less sensitive to serine/glycine depletion in medium containing physiological levels of metabolites. The lower requirement for exogenous serine under these culture conditions reflects both increased de novo serine synthesis and the use of hypoxanthine (not present in the standard medium) to support purine synthesis. Limiting serine availability leads to increased uptake of extracellular hypoxanthine, sparing available serine for other pathways such as glutathione synthesis. Taken together these results improve our understanding of serine metabolism in physiologically relevant nutrient conditions and allow us to predict interventions that may enhance the therapeutic response to dietary serine/glycine limitation.

Serine synthesis pathway inhibition cooperates with dietary serine and glycine limitation for cancer therapy.

Many tumour cells show dependence on exogenous serine and dietary serine and glycine starvation can inhibit the growth of these cancers and extend survival in mice. However, numerous mechanisms promote resistance to this therapeutic approach, including enhanced expression of the de novo serine synthesis pathway (SSP) enzymes or activation of oncogenes that drive enhanced serine synthesis. Here we show that inhibition of PHGDH, the first step in the SSP, cooperates with serine and glycine depletion to inhibit one-carbon metabolism and cancer growth. In vitro, inhibition of PHGDH combined with serine starvation leads to a defect in global protein synthesis, which blocks the activation of an ATF-4 response and more broadly impacts the protective stress response to amino acid depletion. In vivo, the combination of diet and inhibitor shows therapeutic efficacy against tumours that are resistant to diet or drug alone, with evidence of reduced one-carbon availability. However, the defect in ATF4-response seen in vitro following complete depletion of available serine is not seen in mice, where dietary serine and glycine depletion and treatment with the PHGDH inhibitor lower but do not eliminate serine. Our results indicate that inhibition of PHGDH will augment the therapeutic efficacy of a serine depleted diet.

Lack of skeletal muscle liver kinase B1 alters gene expression, mitochondrial content, inflammation and oxidative stress without affecting high-fat diet-induced obesity or insulin resistance.

Ad libitum high-fat diet (HFD) induces obesity and skeletal muscle metabolic dysfunction. Liver kinase B1 (LKB1) regulates skeletal muscle metabolism by controlling the AMP-activated protein kinase family, but its importance in regulating muscle gene expression and glucose tolerance in obese mice has not been established. The purpose of this study was to determine how the lack of LKB1 in skeletal muscle (KO) affects gene expression and glucose tolerance in HFD-fed, obese mice. KO and littermate control wild-type (WT) mice were fed a standard diet or HFD for 14 weeks. RNA sequencing, and subsequent analysis were performed to assess mitochondrial content and respiration, inflammatory status, glucose and insulin tolerance, and muscle anabolic signaling. KO did not affect body weight gain on HFD, but heavily impacted mitochondria-, oxidative stress-, and inflammation-related gene expression. Accordingly, mitochondrial protein content and respiration were suppressed while inflammatory signaling and markers of oxidative stress were elevated in obese KO muscles. KO did not affect glucose or insulin tolerance. However, fasting serum insulin and skeletal muscle insulin signaling were higher in the KO mice. Furthermore, decreased muscle fiber size in skmLKB1-KO mice was associated with increased general protein ubiquitination and increased expression of several ubiquitin ligases, but not muscle ring finger 1 or atrogin-1. Taken together, these data suggest that the lack of LKB1 in skeletal muscle does not exacerbate obesity or insulin resistance in mice on a HFD, despite impaired mitochondrial content and function and elevated inflammatory signaling and oxidative stress.

Dynamic ROS Control by TIGAR Regulates the Initiation and Progression of Pancreatic Cancer.

The TIGAR protein has antioxidant activity that supports intestinal tissue repair and adenoma development. Using a pancreatic ductal adenocarcinoma (PDAC) model, we show that reactive oxygen species (ROS) regulation by TIGAR supports premalignant tumor initiation while restricting metastasis. Increased ROS in PDAC cells drives a phenotypic switch that increases migration, invasion, and metastatic capacity. This switch is dependent on increased activation of MAPK signaling and can be reverted by antioxidant treatment. In mouse and human, TIGAR expression is modulated during PDAC development, with higher TIGAR levels in premalignant lesions and lower TIGAR levels in metastasizing tumors. Our study indicates that temporal, dynamic control of ROS underpins full malignant progression and helps to rationalize conflicting reports of pro- and anti-tumor effects of antioxidant treatment.

Cell Clustering Promotes a Metabolic Switch that Supports Metastatic Colonization.

Cancer metastasis depends on cell survival following loss of extracellular matrix attachment and dissemination through the circulation. The metastatic spread can be enhanced by the clustering of detached cancer cells and increased antioxidant defense. Here, we link these responses by describing how cell clustering limits reactive oxygen species (ROS). Loss of attachment causes mitochondrial perturbations and increased ROS production. The formation of cell clusters induces a hypoxic environment that drives hypoxia-inducible factor 1-alpha (Hif1α)-mediated mitophagy, clearing damaged mitochondria and limiting ROS. However, hypoxia and reduced mitochondrial capacity promote dependence on glycolysis for ATP production that is supported by cytosolic reductive metabolism. Preventing this metabolic adaptation or disruption of cell clusters results in ROS accumulation, cell death, and a reduction of metastatic capacity in vivo. Our results provide a mechanistic explanation for the role of cell clustering in supporting survival during extracellular matrix detachment and metastatic spread and may point to targetable vulnerabilities.

ß-Hydroxybutyrate Elicits Favorable Mitochondrial Changes in Skeletal Muscle.

The clinical benefit of ketosis has historically and almost exclusively centered on neurological conditions, lending insight into how ketones alter mitochondrial function in neurons. However, there is a gap in our understanding of how ketones influence mitochondria within skeletal muscle cells. The purpose of this study was to elucidate the specific effects of ß-hydroxybutyrate (ß-HB) on muscle cell mitochondrial physiology. In addition to increased cell viability, murine myotubes displayed beneficial mitochondrial changes evident in reduced H2O2 emission and less mitochondrial fission, which may be a result of a ß-HB-induced reduction in ceramides. Furthermore, muscle from rats in sustained ketosis similarly produced less H2O2 despite an increase in mitochondrial respiration and no apparent change in mitochondrial quantity. In sum, these results indicate a general improvement in muscle cell mitochondrial function when ß-HB is provided as a fuel.

Corrigendum: Modulating the therapeutic response of tumours to dietary serine and glycine starvation.

This corrects the article DOI: 10.1038/nature22056.

Development of an inducible mouse model of iRFP713 to track recombinase activity and tumour development in vivo.

While the use of bioluminescent proteins for molecular imaging is a powerful technology to further our understanding of complex processes, fluorescent labeling with visible light fluorescent proteins such as GFP and RFP suffers from poor tissue penetration and high background autofluorescence. To overcome these limitations, we generated an inducible knock-in mouse model of iRFP713. This model was used to assess Cre activity in a Rosa Cre-ER background and quantify Cre activity upon different tamoxifen treatments in several organs. We also show that iRFP can be readily detected in 3D organoid cultures, FACS analysis and in vivo tumour models. Taken together we demonstrate that iRFP713 is a progressive step in in vivo imaging and analysis that widens the optical imaging window to the near-infrared spectrum, thereby allowing deeper tissue penetration, quicker image acquisition without the need to inject substrates and a better signal to background ratio in genetically engineered mouse models (GEMMs).

Modulating the therapeutic response of tumours to dietary serine and glycine starvation.

The non-essential amino acids serine and glycine are used in multiple anabolic processes that support cancer cell growth and proliferation (reviewed in ref. 1). While some cancer cells upregulate de novo serine synthesis, many others rely on exogenous serine for optimal growth. Restriction of dietary serine and glycine can reduce tumour growth in xenograft and allograft models. Here we show that this observation translates into more clinically relevant autochthonous tumours in genetically engineered mouse models of intestinal cancer (driven by Apc inactivation) or lymphoma (driven by Myc activation). The increased survival following dietary restriction of serine and glycine in these models was further improved by antagonizing the anti-oxidant response. Disruption of mitochondrial oxidative phosphorylation (using biguanides) led to a complex response that could improve or impede the anti-tumour effect of serine and glycine starvation. Notably, Kras-driven mouse models of pancreatic and intestinal cancers were less responsive to depletion of serine and glycine, reflecting an ability of activated Kras to increase the expression of enzymes that are part of the serine synthesis pathway and thus promote de novo serine synthesis.

Opposing effects of TIGAR- and RAC1-derived ROS on Wnt-driven proliferation in the mouse intestine.

Reactive oxygen species (ROS) participate in numerous cell responses, including proliferation, DNA damage, and cell death. Based on these disparate activities, both promotion and inhibition of ROS have been proposed for cancer therapy. However, how the ROS response is determined is not clear. We examined the activities of ROS in a model of Apc deletion, where loss of the Wnt target gene Myc both rescues APC loss and prevents ROS accumulation. Following APC loss, Myc has been shown to up-regulate RAC1 to promote proliferative ROS through NADPH oxidase (NOX). However, APC loss also increased the expression of TIGAR, which functions to limit ROS. To explore this paradox, we used three-dimensional (3D) cultures and in vivo models to show that deletion of TIGAR increased ROS damage and inhibited proliferation. These responses were suppressed by limiting damaging ROS but enhanced by lowering proproliferative NOX-derived ROS. Despite having opposing effects on ROS levels, loss of TIGAR and RAC1 cooperated to suppress intestinal proliferation following APC loss. Our results indicate that the pro- and anti-proliferative effects of ROS can be independently modulated in the same cell, with two key targets in the Wnt pathway functioning to integrate the different ROS signals for optimal cell proliferation.

iRFP is a real time marker for transformation based assays in high content screening.

Anchorage independent growth is one of the hallmarks of oncogenic transformation. Here we show that infrared fluorescent protein (iRFP) based assays allow accurate and unbiased determination of colony formation and anchorage independent growth over time. This protocol is particularly compatible with high throughput systems, in contrast to traditional methods which are often labor-intensive, subjective to bias and do not allow further analysis using the same cells. Transformation in a single layer soft agar assay could be documented as early as 2 to 3 days in a 96 well format, which can be easily combined with standard transfection, infection and compound screening setups to allow for high throughput screening to identify therapeutic targets.

TIGAR, TIGAR, burning bright.

Cancers cells shift their metabolism towards glycolysis in order to help them support the biosynthetic demands necessary to sustain cell proliferation and growth, adapt to stress and avoid excessive reactive oxygen species (ROS) accumulation. While the p53 tumor suppressor protein is known to inhibit cell growth by inducing apoptosis, senescence and cell cycle arrest, recent studies have found that p53 is also able to influence cell metabolism. TIGAR is a p53 target that functions as a fructose-2,6-bisphosphatase, thereby lowering glycolytic flux and promoting antioxidant functions. By protecting cells from oxidative stress, TIGAR may mediate some of the tumor suppressor activity of p53 but could also contribute to tumorigenesis. Here we discuss the activities of TIGAR described so far, and the potential consequences of TIGAR expression on normal and tumor cells.

Metabolic regulation by p53 family members.

The function of p53 is best understood in response to genotoxic stress, but increasing evidence suggests that p53 also plays a key role in the regulation of metabolic homeostasis. p53 and its family members directly influence various metabolic pathways, enabling cells to respond to metabolic stress. These functions are likely to be important for restraining the development of cancer but could also have a profound effect on the development of metabolic diseases, including diabetes. A better understanding of the metabolic functions of p53 family members may aid in the identification of therapeutic targets and reveal novel uses for p53-modulating drugs.

TIGAR is required for efficient intestinal regeneration and tumorigenesis.

Regulation of metabolic pathways plays an important role in controlling cell growth, proliferation, and survival. TIGAR acts as a fructose-2,6-bisphosphatase, potentially promoting the pentose phosphate pathway to produce NADPH for antioxidant function and ribose-5-phosphate for nucleotide synthesis. The functions of TIGAR were dispensable for normal growth and development in mice but played a key role in allowing intestinal regeneration in vivo and in ex vivo cultures, where growth defects due to lack of TIGAR were rescued by ROS scavengers and nucleosides. In a mouse intestinal adenoma model, TIGAR deficiency decreased tumor burden and increased survival, while elevated expression of TIGAR in human colon tumors suggested that deregulated TIGAR supports cancer progression. Our study demonstrates the importance of TIGAR in regulating metabolism for regeneration and cancer development and identifies TIGAR as a potential therapeutic target in diseases such as ulcerative colitis and intestinal cancer.

Mitochondrial localization of TIGAR under hypoxia stimulates HK2 and lowers ROS and cell death.

The p53-inducible protein TIGAR (Tp53-induced Glycolysis and Apoptosis Regulator) functions as a fructose-2,6-bisphosphatase (Fru-2,6-BPase), and through promotion of the pentose phosphate pathway, increases NADPH production to help limit reactive oxygen species (ROS). Here, we show that under hypoxia, a fraction of TIGAR protein relocalized to mitochondria and formed a complex with hexokinase 2 (HK2), resulting in an increase in HK2 activity. Mitochondrial localization of TIGAR depended on mitochondrial HK2 and hypoxia-inducible factor 1 (HIF1α) activity. The ability of TIGAR to function as a Fru-2,6-BPase was independent of HK2 binding and mitochondrial localization, although both of these activities can contribute to the full activity of TIGAR in limiting mitochondrial ROS levels and protecting from cell death.