Three-dimensional environment sensitizes pancreatic cancer cells to the anti-proliferative effect of budesonide by reprogramming energy metabolism.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer with an aggressive metastatic phenotype and very poor clinical prognosis. Interestingly, a lower occurrence of PDAC has been described in individuals with severe and long-standing asthma. Here we explored the potential link between PDAC and the glucocorticoid (GC) budesonide, a first-line therapy to treat asthma. METHODS: We tested the effect of budesonide and the classical GCs on the morphology, proliferation, migration and invasiveness of patient-derived PDAC cells and pancreatic cancer cell lines, using 2D and 3D cultures in vitro. Furthermore, a xenograft model was used to investigate the effect of budesonide on PDAC tumor growth in vivo. Finally, we combined genome-wide transcriptome analysis with genetic and pharmacological approaches to explore the mechanisms underlying budesonide activities in the different environmental conditions. RESULTS: We found that in 2D culture settings, high micromolar concentrations of budesonide reduced the mesenchymal invasive/migrating features of PDAC cells, without affecting proliferation or survival. This activity was specific and independent of the Glucocorticoid Receptor (GR). Conversely, in a more physiological 3D environment, low nanomolar concentrations of budesonide strongly reduced PDAC cell proliferation in a GR-dependent manner. Accordingly, we found that budesonide reduced PDAC tumor growth in vivo. Mechanistically, we demonstrated that the 3D environment drives the cells towards a general metabolic reprogramming involving protein, lipid, and energy metabolism (e.g., increased glycolysis dependency). This metabolic change sensitizes PDAC cells to the anti-proliferative effect of budesonide, which instead induces opposite changes (e.g., increased mitochondrial oxidative phosphorylation). Finally, we provide evidence that budesonide inhibits PDAC growth, at least in part, through the tumor suppressor CDKN1C/p57Kip2. CONCLUSIONS: Collectively, our study reveals that the microenvironment influences the susceptibility of PDAC cells to GCs and provides unprecedented evidence for the anti-proliferative activity of budesonide on PDAC cells in 3D conditions, in vitro and in vivo. Our findings may explain, at least in part, the reason for the lower occurrence of pancreatic cancer in asthmatic patients and suggest a potential suitability of budesonide for clinical trials as a therapeutic approach to fight pancreatic cancer.

Budesonide Analogues Preserve Stem Cell Pluripotency and Delay 3D Gastruloid Development.

Small molecules that can modulate or stabilize cell-cell interactions are valuable tools for investigating the impact of collective cell behavior on various biological processes such as development/morphogenesis, tissue regeneration and cancer progression. Recently, we showed that budesonide, a glucocorticoid widely used as an anti-asthmatic drug, is a potent regulator of stem cell pluripotency. Here we tested the effect of different budesonide derivatives and identified CHD-030498 as a more effective analogue of budesonide. CHD-030498 was able to prevent stem cell pluripotency exit in different cell-based models, including embryonic stem-to-mesenchymal transition, spontaneous differentiation and 3D gastruloid development, and at lower doses compared to budesonide.

Stabilization of cell-cell adhesions prevents symmetry breaking and locks in pluripotency in 3D gastruloids.

3D embryonic stem cell (ESC) aggregates self-organize into embryo-like structures named gastruloids that recapitulate the axial organization of post-implantation embryos. Crucial in this process is the symmetry-breaking event that leads to the emergence of asymmetry and spatially ordered structures from homogeneous cell aggregates. Here, we show that budesonide, a glucocorticoid drug widely used to treat asthma, prevents ESC aggregates to break symmetry. Mechanistically, the effect of budesonide is glucocorticoid receptor independent. RNA sequencing and lineage fate analysis reveal that budesonide counteracts exit from pluripotency and modifies the expression of a large set of genes associated with cell migration, A-P axis formation, and WNT signaling. This correlates with reduced phenotypic and molecular cell heterogeneity, persistence of E-CADHERIN at the cell-cell interface, and cell aggregate compaction. Our findings reveal that cell-cell adhesion properties control symmetry breaking and cell fate transition in 3D gastruloids and suggest a potential adverse effect of budesonide on embryo development.

Capturing Transitional Pluripotency through Proline Metabolism.

In this paper, we summarize the current knowledge of the role of proline metabolism in the control of the identity of Embryonic Stem Cells (ESCs). An imbalance in proline metabolism shifts mouse ESCs toward a stable naïve-to-primed intermediate state of pluripotency. Proline-induced cells (PiCs), also named primitive ectoderm-like cells (EPLs), are phenotypically metastable, a trait linked to a rapid and reversible relocalization of E-cadherin from the plasma membrane to intracellular membrane compartments. The ESC-to-PiC transition relies on the activation of Erk and Tgfß/Activin signaling pathways and is associated with extensive remodeling of the transcriptome, metabolome and epigenome. PiCs maintain several properties of naïve pluripotency (teratoma formation, blastocyst colonization and 3D gastruloid development) and acquire a few traits of primed cells (flat-shaped colony morphology, aerobic glycolysis metabolism and competence for primordial germ cell fate). Overall, the molecular and phenotypic features of PiCs resemble those of an early-primed state of pluripotency, providing a robust model to study the role of metabolic perturbations in pluripotency and cell fate decisions.

The Multifaceted Roles of Proline in Cell Behavior.

Herein, we review the multifaceted roles of proline in cell biology. This peculiar cyclic imino acid is: (i) A main precursor of extracellular collagens (the most abundant human proteins), antimicrobial peptides (involved in innate immunity), salivary proteins (astringency, teeth health) and cornifins (skin permeability); (ii) an energy source for pathogenic bacteria, protozoan parasites, and metastatic cancer cells, which engage in extracellular-protein degradation to invade their host; (iii) an antistress molecule (an osmolyte and chemical chaperone) helpful against various potential harms (UV radiation, drought/salinity, heavy metals, reactive oxygen species); (iv) a neural metabotoxin associated with schizophrenia; (v) a modulator of cell signaling pathways such as the amino acid stress response and extracellular signal-related kinase pathway; (vi) an epigenetic modifier able to promote DNA and histone hypermethylation; (vii) an inducer of proliferation of stem and tumor cells; and (viii) a modulator of cell morphology and migration/invasiveness. We highlight how proline metabolism impacts beneficial tissue regeneration, but also contributes to the progression of devastating pathologies such as fibrosis and metastatic cancer.

Gastruloid Development Competence Discriminates Different States of Pluripotency.

Floating spheroidal aggregates of mouse embryonic stem cells can develop into polarized/elongated organoids, namely gastruloids. We set up a high-performing assay to measure gastruloid formation efficiency (GFE), and found that GFE decreases as pluripotency progresses from naive (GFE ≥ 95%) to primed (GFE = 0) state. Specifically, we show that primed EpiSCs fail to generate proper cell aggregates, while early-primed EpiLCs aggregate but eventually fail to develop into elongated gastruloids. Moreover, we characterized proline-induced cells (PiCs), a LIF-dependent reversible early-primed state of pluripotency, and show that PiCs are able to generate gastruloids (GFE ∼ 50%) and are also competent to differentiate into primordial germ cell-like cells. Thus, we propose the GFE assay as a valuable functional tool to discriminate different states of the pluripotency continuum.

Proline Metabolism in Tumor Growth and Metastatic Progression.

Cancer cells show a formidable capacity to survive under stringent conditions, to elude mechanisms of control, such as apoptosis, and to resist therapy. Cancer cells reprogram their metabolism to support uncontrolled proliferation and metastatic progression. Phenotypic and functional heterogeneity are hallmarks of cancer cells, which endow them with aggressiveness, metastatic capacity, and resistance to therapy. This heterogeneity is regulated by a variety of intrinsic and extrinsic stimuli including those from the tumor microenvironment. Increasing evidence points to a key role for the metabolism of non-essential amino acids in this complex scenario. Here we discuss the impact of proline metabolism in cancer development and progression, with particular emphasis on the enzymes involved in proline synthesis and catabolism, which are linked to pathways of energy, redox, and anaplerosis. In particular, we emphasize how proline availability influences collagen synthesis and maturation and the acquisition of cancer cell plasticity and heterogeneity. Specifically, we propose a model whereby proline availability generates a cycle based on collagen synthesis and degradation, which, in turn, influences the epigenetic landscape and tumor heterogeneity. Therapeutic strategies targeting this metabolic-epigenetic axis hold great promise for the treatment of metastatic cancers.

Collagen Prolyl Hydroxylation-Dependent Metabolic Perturbation Governs Epigenetic Remodeling and Mesenchymal Transition in Pluripotent and Cancer Cells.

Collagen prolyl hydroxylation (CPH), which is catalyzed by prolyl 4-hydroxylase (P4H), is the most prevalent posttranslational modification in humans and requires vitamin C (VitC). Here, we demonstrate that CPH acts as an epigenetic modulator of cell plasticity. Increased CPH induced global DNA/histone methylation in pluripotent stem and tumor cells and promoted cell state transition (CST). Interfering with CPH by either genetic ablation of P4H subunit alpha-2 (P4HA2) or pharmacologic treatment reverted epigenetic changes and antagonized CST. Mechanistically, we suggest that CPH modifies the epigenetic landscape by reducing VitC for DNA and histone demethylases. Repurposed drugs targeting CPH-mediated metabolic perturbation, such as the antiasthmatic budesonide, blocked metastatic dissemination of breast cancer cells in vivo by preventing mesenchymal transition. Our study provides mechanistic insights into how metabolic cues and epigenetic factors integrate to control CST and paves the way for the development of novel antimetastatic strategies. SIGNIFICANCE: A phenotype-based high-throughput screening reveals unforeseen metabolic control of cell plasticity and identifies budesonide as a drug candidate for metastatic cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3235/F1.large.jpg.

Metabolic-Epigenetic Axis in Pluripotent State Transitions.

Cell state transition (CST) occurs during embryo development and in adult life in response to different stimuli and is associated with extensive epigenetic remodeling. Beyond growth factors and signaling pathways, increasing evidence point to a crucial role of metabolic signals in this process. Indeed, since several epigenetic enzymes are sensitive to availability of specific metabolites, fluctuations in their levels may induce the epigenetic changes associated with CST. Here we analyze how fluctuations in metabolites availability influence DNA/chromatin modifications associated with pluripotent stem cell (PSC) transitions. We discuss current studies and focus on the effects of metabolites in the context of naïve to primed transition, PSC differentiation and reprogramming of somatic cells to induced pluripotent stem cells (iPSCs), analyzing their mechanism of action and the causal correlation between metabolites availability and epigenetic alteration.

Vitamin C in Stem Cell Biology: Impact on Extracellular Matrix Homeostasis and Epigenetics.

Transcription factors and signaling molecules are well-known regulators of stem cell identity and behavior; however, increasing evidence indicates that environmental cues contribute to this complex network of stimuli, acting as crucial determinants of stem cell fate. l-Ascorbic acid (vitamin C (VitC)) has gained growing interest for its multiple functions and mechanisms of action, contributing to the homeostasis of normal tissues and organs as well as to tissue regeneration. Here, we review the main functions of VitC and its effects on stem cells, focusing on its activity as cofactor of Fe+2/αKG dioxygenases, which regulate the epigenetic signatures, the redox status, and the extracellular matrix (ECM) composition, depending on the enzymes' subcellular localization. Acting as cofactor of collagen prolyl hydroxylases in the endoplasmic reticulum, VitC regulates ECM/collagen homeostasis and plays a key role in the differentiation of mesenchymal stem cells towards osteoblasts, chondrocytes, and tendons. In the nucleus, VitC enhances the activity of DNA and histone demethylases, improving somatic cell reprogramming and pushing embryonic stem cell towards the naive pluripotent state. The broad spectrum of actions of VitC highlights its relevance for stem cell biology in both physiology and disease.

Vitamin C and l-Proline Antagonistic Effects Capture Alternative States in the Pluripotency Continuum.

Metabolites and cofactors are emerging as key regulators of cell plasticity and reprogramming, and their role in the control of pluripotency is just being discovered. Here we provide unprecedented evidence that embryonic stem cell (ESC) pluripotency relies on the relative levels of two physiological metabolites, namely ascorbic acid (vitamin C, VitC) and l-proline (l-Pro), which affect global DNA methylation, transcriptional profile, and energy metabolism. Specifically, while a high VitC/l-Pro ratio drives ESCs toward a naive state, the opposite condition (l-Pro excess) captures a fully reversible early primed pluripotent state, which depends on autocrine fibroblast growth factor and transforming growth factor ß signaling pathways. Our findings highlight the pivotal role of metabolites availability in controlling the pluripotency continuum from naive to primed states.

Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency.

Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/ß-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo.

Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro--correlation between contraction force and electrophysiology.

Cardiomyocytes from human pluripotent stem cells (hPSC-CM) have many potential applications in disease modelling and drug target discovery but their phenotypic similarity to early fetal stages of cardiac development limits their applicability. In this study we compared contraction stresses of hPSC-CM to 2nd trimester human fetal derived cardiomyocytes (hFetal-CM) by imaging displacement of fluorescent beads by single contracting hPSC-CM, aligned by microcontact-printing on polyacrylamide gels. hPSC-CM showed distinctly lower contraction stress than cardiomyocytes isolated from hFetal-CM. To improve maturation of hPSC-CM in vitro we made use of commercial media optimized for cardiomyocyte maturation, which promoted significantly higher contraction stress in hPSC-compared with hFetal-CM. Accordingly, other features of cardiomyocyte maturation were observed, most strikingly increased upstroke velocities and action potential amplitudes, lower resting membrane potentials, improved sarcomeric organization and alterations in cardiac-specific gene expression. Performing contraction force and electrophysiology measurements on individual cardiomyocytes revealed strong correlations between an increase in contraction force and a rise of the upstroke velocity and action potential amplitude and with a decrease in the resting membrane potential. We showed that under standard differentiation conditions hPSC-CM display lower contractile force than primary hFetal-CM and identified conditions under which a commercially available culture medium could induce molecular, morphological and functional maturation of hPSC-CM in vitro. These results are an important contribution for full implementation of hPSC-CM in cardiac disease modelling and drug discovery.

Recessive cardiac phenotypes in induced pluripotent stem cell models of Jervell and Lange-Nielsen syndrome: disease mechanisms and pharmacological rescue.

Jervell and Lange-Nielsen syndrome (JLNS) is one of the most severe life-threatening cardiac arrhythmias. Patients display delayed cardiac repolarization, associated high risk of sudden death due to ventricular tachycardia, and congenital bilateral deafness. In contrast to the autosomal dominant forms of long QT syndrome, JLNS is a recessive trait, resulting from homozygous (or compound heterozygous) mutations in KCNQ1 or KCNE1. These genes encode the α and ß subunits, respectively, of the ion channel conducting the slow component of the delayed rectifier K(+) current, IKs. We used complementary approaches, reprogramming patient cells and genetic engineering, to generate human induced pluripotent stem cell (hiPSC) models of JLNS, covering splice site (c.478-2A>T) and missense (c.1781G>A) mutations, the two major classes of JLNS-causing defects in KCNQ1. Electrophysiological comparison of hiPSC-derived cardiomyocytes (CMs) from homozygous JLNS, heterozygous, and wild-type lines recapitulated the typical and severe features of JLNS, including pronounced action and field potential prolongation and severe reduction or absence of IKs. We show that this phenotype had distinct underlying molecular mechanisms in the two sets of cell lines: the previously unidentified c.478-2A>T mutation was amorphic and gave rise to a strictly recessive phenotype in JLNS-CMs, whereas the missense c.1781G>A lesion caused a gene dosage-dependent channel reduction at the cell membrane. Moreover, adrenergic stimulation caused action potential prolongation specifically in JLNS-CMs. Furthermore, sensitivity to proarrhythmic drugs was strongly enhanced in JLNS-CMs but could be pharmacologically corrected. Our data provide mechanistic insight into distinct classes of JLNS-causing mutations and demonstrate the potential of hiPSC-CMs in drug evaluation.

Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome.

Patient-specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long-QT syndromes (LQTS). To study the LQT2-associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (IKr) conducted by the HERG channel and the action potential (AP) duration in iPSC-derived cardiomyocytes (CMs). Introduction of the same mutation reduced IKr and prolonged the AP duration in hESC-derived CMs. Further characterization of N996I-HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.

The G-protein-coupled receptor APJ is expressed in the second heart field and regulates Cerberus-Baf60c axis in embryonic stem cell cardiomyogenesis.

AIMS: Mammalian cardiomyogenesis occurs through a multistep process that requires a complex network of tightly regulated extracellular signals, which integrate with the genetic and epigenetic machinery to maintain, expand, and regulate the differentiation of cardiac progenitor cells. Pluripotent embryonic stem cells (ESCs) recapitulate many aspects of development, and have provided an excellent opportunity to dissect the molecular mechanisms underlying cardiomyogenesis, which is still incompletely defined. METHODS AND RESULTS: We provide new in vivo evidence that the G-protein-coupled receptor angiotensin receptor-like 1 (Apj) is expressed in the mesodermal cells of the second heart field, a population of cardiac progenitors that give rise to a major part of the definitive heart. By combining loss-and-gain of function studies in mouse ESCs, we show that Apj (i) controls the balance between proliferation and cardiovascular differentiation, (ii) regulates the Nodal/Bone Morphogenetic Protein antagonist Cerberus and the Baf60c/Smarcd3 subunit of the Brg1/Brm-associated factors (BAF) chromatin-remodelling complex. CONCLUSION: We propose a model in which Apj controls a regulatory Cerberus-Baf60c pathway in pluripotent stem cell cardiomyogenesis, and speculate that this regulatory circuit may regulate cardiac progenitor cell behaviour.

Temporal proteomic profiling of embryonic stem cell secretome during cardiac and neural differentiation.

During recent years, increased efforts have focused on elucidating the pluripotency and self-renewal of stem cells. Differentiation towards the different lineages has attracted significant attention given the potential use of stem cells in regenerative medicine. Embryonic stem cell differentiation is a complex process coordinated by strictly regulated extracellular signals that act in an autocrine and/or paracrine manner. Through secreted molecules, stem cells affect local niche biology and influence the cross-talking with the surrounding tissues. Emerging evidence supports the hypothesis that fundamental cell functions, including proliferation and differentiation, are strictly regulated by the complex set of molecules secreted from cells. The understanding of this molecular language could largely increase our knowledge on pathways regulating stem cell differentiation. Here, we have used a proteomics platform to investigate the profile of proteins secreted during differentiation of murine embryonic stem cells. We have followed the dynamics of protein secretion by comparing the secretomes at different time points of murine embryonic stem cell cardiac and neural differentiation. In addition to previously reported molecules, we have identified many secreted proteins not described so far as released from embryonic stem cells nor shown to be differentially released during the process of cardiomyogenesis and neurogenesis.

G protein-coupled receptor APJ and its ligand apelin act downstream of Cripto to specify embryonic stem cells toward the cardiac lineage through extracellular signal-regulated kinase/p70S6 kinase signaling pathway.

RATIONALE: Pluripotent stem cells represent a powerful model system to study the early steps of cardiac specification for which the molecular control is largely unknown. The EGF-CFC (epidermal growth factor-Cripto/FRL-1/Cryptic) Cripto protein is essential for cardiac myogenesis in embryonic stem cells (ESCs). OBJECTIVE: Here, we study the role of apelin and its G protein-coupled receptor, APJ, as downstream targets of Cripto both in vivo and in ESC differentiation. METHODS AND RESULTS: Gain-of-function experiments show that APJ suppresses neuronal differentiation and restores the cardiac program in Cripto(-/-) ESCs. Loss-of-function experiments point for a central role for APJ/apelin in the gene regulatory cascade promoting cardiac specification and differentiation in ESCs. Remarkably, we show for the first time that apelin promotes mammalian cardiomyogenesis via activation of mitogen-activated protein kinase/p70S6 through coupling to a Go/Gi protein. CONCLUSIONS: Together our data provide evidence for a previously unrecognized function of APJ/apelin in the Cripto signaling pathway governing mesoderm patterning and cardiac specification in mammals.