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.

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.

Long non-coding RNA in stem cell pluripotency and lineage commitment: functions and evolutionary conservation.

LncRNAs have recently emerged as new and fundamental transcriptional and post-transcriptional regulators acting at multiple levels of gene expression. Indeed, lncRNAs participate in a wide variety of stem cell and developmental processes, acting in cis and/or in trans in the nuclear and/or in the cytoplasmic compartments, and generating an intricate network of interactions with RNAs, enhancers, and chromatin-modifier complexes. Given the versatility of these molecules to operate in different subcellular compartments, via different modes of action and with different target specificity, the interest in this research field is rapidly growing. Here, we review recent progress in defining the functional role of lncRNAs in stem cell biology with a specific focus on the underlying mechanisms. We also discuss recent findings on a new family of evolutionary conserved lncRNAs transcribed from ultraconserved elements, which show perfect conservation between human, mouse, and rat genomes, and that are emerging as new player in this complex scenario.

LncRNAs and PRC2: Coupled Partners in Embryonic Stem Cells.

The power of embryonic stem cells (ESCs) lies in their ability to self-renew and differentiate. Behind these two unique capabilities is a fine-tuned molecular network that shapes the genetic, epigenetic, and epitranscriptomic ESC plasticity. Although RNA has been shown to be functionally important in only a small minority of long non-coding RNA genes, a growing body of evidence has highlighted the pivotal and intricate role of lncRNAs in chromatin remodeling. Due to their multifaceted nature, lncRNAs interact with DNA, RNA, and proteins, and are emerging as new modulators of extensive gene expression programs through their participation in ESC-specific regulatory circuitries. Here, we review the tight cooperation between lncRNAs and Polycomb repressive complex 2 (PRC2), which is intimately involved in determining and maintaining the ESC epigenetic landscape. The lncRNA-PRC2 partnership is fundamental in securing the fully pluripotent state of ESCs, which must be primed to differentiate properly. We also reflect on the advantages brought to this field of research by the advent of single-cell analysis.

An Ultraconserved Element Containing lncRNA Preserves Transcriptional Dynamics and Maintains ESC Self-Renewal.

Ultraconserved elements (UCEs) show the peculiar feature to retain extended perfect sequence identity among human, mouse, and rat genomes. Most of them are transcribed and represent a new family of long non-coding RNAs (lncRNAs), the transcribed UCEs (T-UCEs). Despite their involvement in human cancer, the physiological role of T-UCEs is still unknown. Here, we identify a lncRNA containing the uc.170+, named T-UCstem1, and provide in vitro and in vivo evidence that it plays essential roles in embryonic stem cells (ESCs) by modulating cytoplasmic miRNA levels and preserving transcriptional dynamics. Specifically, while T-UCstem1::miR-9 cytoplasmic interplay regulates ESC proliferation by reducing miR-9 levels, nuclear T-UCstem1 maintains ESC self-renewal and transcriptional identity by stabilizing polycomb repressive complex 2 on bivalent domains. Altogether, our findings provide unprecedented evidence that T-UCEs regulate physiological cellular functions and point to an essential role of T-UCstem1 in preserving ESC identity.

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.

Control of embryonic stem cell metastability by L-proline catabolism.

The molecular mechanisms controlling mouse embryonic stem cell (ESC) metastability, i.e. their capacity to fluctuate between different states of pluripotency, are not fully resolved. We developed and used a novel automation platform, the Cell(maker), to screen a library of metabolites on two ESC-based phenotypic assays (i.e. proliferation and colony phenotype) and identified two metabolically related amino acids, namely l-proline (l-Pro) and l-ornithine (l-Orn), as key regulators of ESC metastability. Both compounds, but mainly l-Pro, force ESCs toward a novel epiblast stem cell (EpiSC)-like state, in a dose- and time-dependent manner. Unlike EpiSCs, l-Pro-induced cells (PiCs) contribute to chimeric embryos and rely on leukemia inhibitor factor (LIF) to self-renew. Furthermore, PiCs revert to ESCs or differentiate randomly upon removal of either l-Pro or LIF, respectively. Remarkably, PiC generation depends on both l-Pro metabolism (uptake and oxidation) and Fgf5 induction, and is strongly counteracted by antioxidants, mainly l-ascorbic acid (vitamin C, Vc). ESCs ↔ PiCs phenotypic transition thus represents a previously undefined dynamic equilibrium between pluripotent states, which can be unbalanced either toward an EpiSC-like or an ESC phenotype by l-Pro/l-Orn or Vc treatments, respectively. All together, our data provide evidence that ESC metastability can be regulated at a metabolic level.