The glutathione S-transferase Gstt1 drives survival and dissemination in metastases.

Identifying the adaptive mechanisms of metastatic cancer cells remains an elusive question in the treatment of metastatic disease, particularly in pancreatic cancer (pancreatic adenocarcinoma, PDA). A loss-of-function shRNA targeted screen in metastatic-derived cells identified Gstt1, a member of the glutathione S-transferase superfamily, as uniquely required for dissemination and metastasis, but dispensable for primary tumour growth. Gstt1 is expressed in latent disseminated tumour cells (DTCs), is retained within a subpopulation of slow-cycling cells within existing metastases, and its inhibition leads to complete regression of macrometastatic tumours. This distinct Gstt1high population is highly metastatic and retains slow-cycling phenotypes, epithelial-mesenchymal transition features and DTC characteristics compared to the Gstt1low population. Mechanistic studies indicate that in this subset of cancer cells, Gstt1 maintains metastases by binding and glutathione-modifying intracellular fibronectin, in turn promoting its secretion and deposition into the metastatic microenvironment. We identified Gstt1 as a mediator of metastasis, highlighting the importance of heterogeneity and its influence on the metastatic tumour microenvironment.

Osmolar Modulation Drives Reversible Cell Cycle Exit and Human Pluripotent Cell Differentiation via NF-κВ and WNT Signaling.

Terminally differentiated cells are commonly regarded as the most stable cell state in adult organisms, characterized by growth arrest while fulfilling their specialized functions. A better understanding of the mechanisms involved in promoting cell cycle exit will improve the ability to differentiate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, it demonstrates that a hyperosmolar environment enforces a protective p53-independent quiescent state in immature hepatoma cells and in pluripotent stem cell-derived models of human hepatocytes and endothelial cells. Prolonged culture in hyperosmolar conditions stimulates changes in gene expression promoting functional cell maturation. Interestingly, hyperosmolar conditions do not only trigger growth arrest and cellular maturation but are also necessary to maintain this maturated state, as switching back to plasma osmolarity reverses the changes in expression of maturation and proliferative markers. Transcriptome analysis revealed sequential stages of osmolarity-regulated growth arrest followed by cell maturation, mediated by activation of NF-κВ, and repression of WNT signaling, respectively. This study reveals that a modulated increase in osmolarity serves as a biochemical signal to promote long-term growth arrest and cellular maturation into different lineages, providing a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely.

Inhibition of the proline metabolism rate-limiting enzyme P5CS allows proliferation of glutamine-restricted cancer cells.

Glutamine is a critical metabolite for rapidly proliferating cells as it is used for the synthesis of key metabolites necessary for cell growth and proliferation. Glutamine metabolism has been proposed as a therapeutic target in cancer and several chemical inhibitors are in development or in clinical trials. How cells subsist when glutamine is limiting is poorly understood. Here, using an unbiased screen, we identify ALDH18A1, which encodes P5CS, the rate-limiting enzyme in the proline biosynthetic pathway, as a gene that cells can downregulate in response to glutamine starvation. Notably, P5CS downregulation promotes de novo glutamine synthesis, highlighting a previously unrecognized metabolic plasticity of cancer cells. The glutamate conserved from reducing proline synthesis allows cells to produce the key metabolites necessary for cell survival and proliferation under glutamine-restricted conditions. Our findings reveal an adaptive pathway that cancer cells acquire under nutrient stress, identifying proline biosynthesis as a previously unrecognized major consumer of glutamate, a pathway that could be exploited for developing effective metabolism-driven anticancer therapies.

Systematic transcriptome-based comparison of cellular adaptive stress response activation networks in hepatic stem cell-derived progeny and primary human hepatocytes.

Various adaptive cellular stress response pathways are critical in the pathophysiology of liver disease and drug-induced liver injury. Human-induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (HLCs) provide a promising tool to study cellular stress response pathways, but in this context there is limited insight on how HLCs compare to other in vitro liver models. Here, we systematically compared the transcriptomic profiles upon chemical activation in HLCs, hiPSC, primary human hepatocytes (PHH) and HepG2 liver cancer cells. We used targeted RNA-sequencing to map concentration transcriptional response using benchmark concentration modeling for the various stress responses in the different test systems. We found that HLCs are very sensitive towards oxidative stress and inflammation conditions as corresponding genes were activated at over 3 fold lower concentrations of the corresponding pathway inducing compounds as compared to PHH. PHH were the most sensitive model when studying UPR related effects. Due to the non-proliferative nature of PHH and HLCs, these do not pose a good/sensitive model to pick up DNA damage responses, while hiPSC and HepG2 were more sensitive in these conditions. We envision that this study contributes to a better understanding on how HLCs can contribute to the assessment of cell physiological stress response activation to predict hepatotoxic events.

A unique subset of glycolytic tumour-propagating cells drives squamous cell carcinoma.

Head and neck squamous cell carcinoma (SCC) remains among the most aggressive human cancers. Tumour progression and aggressiveness in SCC are largely driven by tumour-propagating cells (TPCs). Aerobic glycolysis, also known as the Warburg effect, is a characteristic of many cancers; however, whether this adaptation is functionally important in SCC, and at which stage, remains poorly understood. Here, we show that the NAD+-dependent histone deacetylase sirtuin 6 is a robust tumour suppressor in SCC, acting as a modulator of glycolysis in these tumours. Remarkably, rather than a late adaptation, we find enhanced glycolysis specifically in TPCs. More importantly, using single-cell RNA sequencing of TPCs, we identify a subset of TPCs with higher glycolysis and enhanced pentose phosphate pathway and glutathione metabolism, characteristics that are strongly associated with a better antioxidant response. Together, our studies uncover enhanced glycolysis as a main driver in SCC, and, more importantly, identify a subset of TPCs as the cell of origin for the Warburg effect, defining metabolism as a key feature of intra-tumour heterogeneity.

PU.1 drives specification of pluripotent stem cell-derived endothelial cells to LSEC-like cells.

To date, there is no representative in vitro model for liver sinusoidal endothelial cells (LSECs), as primary LSECs dedifferentiate very fast in culture and no combination of cytokines or growth factors can induce an LSEC fate in (pluripotent stem cell (PSC)-derived) endothelial cells (ECs). Furthermore, the transcriptional programmes driving an LSEC fate have not yet been described. Here, we first present a computational workflow (CenTFinder) that can identify transcription factors (TFs) that are crucial for modulating pathways involved in cell lineage specification. Using CenTFinder, we identified several novel LSEC-specific protein markers, such as FCN2 and FCN3, which were validated by analysis of previously published single-cell RNAseq data. We also identified PU.1 (encoded by the SPI1 gene) as a major regulator of LSEC-specific immune functions. We show that SPI1 overexpression (combined with the general EC TF ETV2) in human PSCs induces ECs with an LSEC-like phenotype. The ETV2-SPI1-ECs display increased expression of LSEC markers, such as CD32B and MRC1, as well as several of the proposed novel markers. More importantly, ETV2-SPI1-ECs acquire LSEC functions, including uptake of FSA-FITC, as well as labelled IgG. In conclusion, we present the CenTFinder computational tool to identify key regulatory TFs within specific pathways, in this work pathways of lineage specification, and we demonstrate its use by the identification and validation of PU.1 as a master regulator for LSEC fating.

Nuclear metabolism and the regulation of the epigenome.

Cellular metabolism has emerged as a major biological node governing cellular behaviour. Metabolic pathways fuel cellular energy needs, providing basic chemical molecules to sustain cellular homeostasis, proliferation and function. Changes in nutrient consumption or availability therefore can result in complete reprogramming of cellular metabolism towards stabilizing core metabolite pools, such as ATP, S-adenosyl methionine, acetyl-CoA, NAD/NADP and α-ketoglutarate. Because these metabolites underlie a variety of essential metabolic reactions, metabolism has evolved to operate in separate subcellular compartments through diversification of metabolic enzyme complexes, oscillating metabolic activity and physical separation of metabolite pools. Given that these same core metabolites are also consumed by chromatin modifiers in the establishment of epigenetic signatures, metabolite consumption on and release from chromatin directly influence cellular metabolism and gene expression. In this Review, we highlight recent studies describing the mechanisms determining nuclear metabolism and governing the redistribution of metabolites between the nuclear and non-nuclear compartments.

Amino acid levels determine metabolism and CYP450 function of hepatocytes and hepatoma cell lines.

Predicting drug-induced liver injury in a preclinical setting remains challenging, as cultured primary human hepatocytes (PHHs), pluripotent stem cell-derived hepatocyte-like cells (HLCs), and hepatoma cells exhibit poor drug biotransformation capacity. We here demonstrate that hepatic functionality depends more on cellular metabolism and extracellular nutrients than on developmental regulators. Specifically, we demonstrate that increasing extracellular amino acids beyond the nutritional need of HLCs and HepG2 cells induces glucose independence, mitochondrial function, and the acquisition of a transcriptional profile that is closer to PHHs. Moreover, we show that these high levels of amino acids are sufficient to drive HLC and HepG2 drug biotransformation and liver-toxin sensitivity to levels similar to those in PHHs. In conclusion, we provide data indicating that extracellular nutrient levels represent a major determinant of cellular maturity and can be utilized to guide stem cell differentiation to the hepatic lineage.

Breast cancer cells rely on environmental pyruvate to shape the metastatic niche.

The extracellular matrix is a major component of the local environment-that is, the niche-that determines cell behaviour1. During metastatic growth, cancer cells shape the extracellular matrix of the metastatic niche by hydroxylating collagen to promote their own metastatic growth2,3. However, only particular nutrients might support the ability of cancer cells to hydroxylate collagen, because nutrients dictate which enzymatic reactions are active in cancer cells4,5. Here we show that breast cancer cells rely on the nutrient pyruvate to drive collagen-based remodelling of the extracellular matrix in the lung metastatic niche. Specifically, we discovered that pyruvate uptake induces the production of α-ketoglutarate. This metabolite in turn activates collagen hydroxylation by increasing the activity of the enzyme collagen prolyl-4-hydroxylase (P4HA). Inhibition of pyruvate metabolism was sufficient to impair collagen hydroxylation and consequently the growth of breast-cancer-derived lung metastases in different mouse models. In summary, we provide a mechanistic understanding of the link between collagen remodelling and the nutrient environment in the metastatic niche.

Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity.

Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.

SOX10 Single Transcription Factor-Based Fast and Efficient Generation of Oligodendrocytes from Human Pluripotent Stem Cells.

Scarce access to primary samples and lack of efficient protocols to generate oligodendrocytes (OLs) from human pluripotent stem cells (hPSCs) are hampering our understanding of OL biology and the development of novel therapies. Here, we demonstrate that overexpression of the transcription factor SOX10 is sufficient to generate surface antigen O4-positive (O4+) and myelin basic protein-positive OLs from hPSCs in only 22 days, including from patients with multiple sclerosis or amyotrophic lateral sclerosis. The SOX10-induced O4+ population resembles primary human OLs at the transcriptome level and can myelinate neurons in vivo. Using in vitro OL-neuron co-cultures, myelination of neurons by OLs can also be demonstrated, which can be adapted to a high-throughput screening format to test the response of pro-myelinating drugs. In conclusion, we provide an approach to generate OLs in a very rapid and efficient manner, which can be used for disease modeling, drug discovery efforts, and potentially for therapeutic OL transplantation.

Dynamic regulation of EZH2 from HPSc to hepatocyte-like cell fate.

Currently, drug metabolization and toxicity studies rely on the use of primary human hepatocytes and hepatoma cell lines, which both have conceivable limitations. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are an alternative and valuable source of hepatocytes that can overcome these limitations. EZH2 (enhancer of zeste homolog 2), a transcriptional repressor of the polycomb repressive complex 2 (PRC2), may play an important role in hepatocyte development, but its role during in vitro hPSC-HLC differentiation has not yet been assessed. We here demonstrate dynamic regulation of EZH2 during hepatic differentiation of hPSC. To enhance EZH2 expression, we inducibly overexpressed EZH2 between d0 and d8, demonstrating a significant improvement in definitive endoderm formation, and improved generation of HLCs. Despite induction of EZH2 overexpression until d8, EZH2 transcript and protein levels decreased from d4 onwards, which might be caused by expression of microRNAs predicted to inhibit EZH2 expression. In conclusion, our studies demonstrate that EZH2 plays a role in endoderm formation and hepatocyte differentiation, but its expression is tightly post-transcriptionally regulated during this process.

HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disorder due to selective loss of motor neurons (MNs). Mutations in the fused in sarcoma (FUS) gene can cause both juvenile and late onset ALS. We generated and characterized induced pluripotent stem cells (iPSCs) from ALS patients with different FUS mutations, as well as from healthy controls. Patient-derived MNs show typical cytoplasmic FUS pathology, hypoexcitability, as well as progressive axonal transport defects. Axonal transport defects are rescued by CRISPR/Cas9-mediated genetic correction of the FUS mutation in patient-derived iPSCs. Moreover, these defects are reproduced by expressing mutant FUS in human embryonic stem cells (hESCs), whereas knockdown of endogenous FUS has no effect, confirming that these pathological changes are mutant FUS dependent. Pharmacological inhibition as well as genetic silencing of histone deacetylase 6 (HDAC6) increase α-tubulin acetylation, endoplasmic reticulum (ER)-mitochondrial overlay, and restore the axonal transport defects in patient-derived MNs.Amyotrophic lateral sclerosis (ALS) leads to selective loss of motor neurons. Using motor neurons derived from induced pluripotent stem cells from patients with ALS and FUS mutations, the authors demonstrate that axonal transport deficits that are observed in these cells can be rescued by HDAC6 inhibition.

Proline metabolism supports metastasis formation and could be inhibited to selectively target metastasizing cancer cells.

Metastases are the leading cause of mortality in patients with cancer. Metastasis formation requires cancer cells to adapt their cellular phenotype. However, how metabolism supports this adaptation of cancer cells is poorly defined. We use 2D versus 3D cultivation to induce a shift in the cellular phenotype of breast cancer cells. We discover that proline catabolism via proline dehydrogenase (Prodh) supports growth of breast cancer cells in 3D culture. Subsequently, we link proline catabolism to in vivo metastasis formation. In particular, we find that PRODH expression and proline catabolism is increased in metastases compared to primary breast cancers of patients and mice. Moreover, inhibiting Prodh is sufficient to impair formation of lung metastases in the orthotopic 4T1 and EMT6.5 mouse models, without adverse effects on healthy tissue and organ function. In conclusion, we discover that Prodh is a potential drug target for inhibiting metastasis formation.

Dual loss of succinate dehydrogenase (SDH) and complex I activity is necessary to recapitulate the metabolic phenotype of SDH mutant tumors.

Mutations in succinate dehydrogenase (SDH) are associated with tumor development and neurodegenerative diseases. Only in tumors, loss of SDH activity is accompanied with the loss of complex I activity. Yet, it remains unknown whether the metabolic phenotype of SDH mutant tumors is driven by loss of complex I function, and whether this contributes to the peculiarity of tumor development versus neurodegeneration. We addressed this question by decoupling loss of SDH and complex I activity in cancer cells and neurons. We found that sole loss of SDH activity was not sufficient to recapitulate the metabolic phenotype of SDH mutant tumors, because it failed to decrease mitochondrial respiration and to activate reductive glutamine metabolism. These metabolic phenotypes were only induced upon the additional loss of complex I activity. Thus, we show that complex I function defines the metabolic differences between SDH mutation associated tumors and neurodegenerative diseases, which could open novel therapeutic options against both diseases.

Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis.

Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo-spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel-specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia-inducible factor (HIF)-1α levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism.

Stem cell-derived hepatocytes: A novel model for hepatitis E virus replication.

BACKGROUND & AIMS: Yearly, approximately 20million people become infected with the hepatitis E virus (HEV) resulting in over 3million cases of acute hepatitis. Although HEV-mediated hepatitis is usually self-limiting, severe cases of fulminant hepatitis as well as chronic infections have been reported, resulting annually in an estimated 60,000 deaths. We studied whether pluripotent stem cell (PSC)-derived hepatocytes, mesodermal and/or neuroprogenitor cells support HEV replication. METHODS: Human PSC were differentiated towards hepatocyte-like cells, mesodermal cells and neuroprogenitors and subsequently infected with HEV. Infection and replication of HEV was analyzed by qRT-PCR, RNA in situ hybridization, negative strand RT-PCR, production of infectious virions and transfection with a transient HEV reporter replicon. RESULTS: PSC-derived hepatocytes supported the complete replication cycle of HEV, as demonstrated by the intracellular presence of positive and negative strand HEV RNA and the production of infectious virions. The replication of the virus in these cells was inhibited by the antiviral drugs ribavirin and interferon-α2b. In contrast to PSC-derived hepatocytes, PSC-derived mesodermal cells and neuroprogenitors only supported HEV replication upon transfection with a HEV subgenomic replicon. CONCLUSION: We demonstrate that PSC can be used to study the hepatotropism of HEV infection. The complete replication cycle of HEV can be recapitulated in infected PSC-derived hepatocytes. By contrast other germ layer cells support intracellular replication but are not infectable with HEV. Thus the early steps in the viral cycle are the main determinant governing HEV tissue tropism. PSC-hepatocytes offer a physiological relevant tool to study the biology of HEV infection and replication and may aid in the design of therapeutic strategies.

Highly proliferative primitive fetal liver hematopoietic stem cells are fueled by oxidative metabolic pathways.

Hematopoietic stem cells (HSCs) in the fetal liver (FL) unlike adult bone marrow (BM) proliferate extensively, posing different metabolic demands. However, metabolic pathways responsible for the production of energy and cellular building blocks in FL HSCs have not been described. Here, we report that FL HSCs use oxygen dependent energy generating pathways significantly more than their BM counterparts. RNA-Seq analysis of E14.5 FL versus BM derived HSCs identified increased expression levels of genes involved in oxidative phosphorylation (OxPhos) and the citric acid cycle (TCA). We demonstrated that FL HSCs contain more mitochondria than BM HSCs, which resulted in increased levels of oxygen consumption and reactive oxygen species (ROS) production. Higher levels of DNA repair and antioxidant pathway gene expression may prevent ROS-mediated (geno)toxicity in FL HSCs. Thus, we here for the first time highlight the underestimated importance of oxygen dependent pathways for generating energy and building blocks in FL HSCs.

Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition.

Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.