Tânia Perestrelo

From gametogenesis and stem cells to cancer: common metabolic themes

BACKGROUND Both pluripotent stem cells (PSCs) and cancer cells have been described as having similar metabolic pathways, most notably a penchant for favoring glycolysis even under aerobiosis, suggesting common themes that might be explored for both stem cell differentiation and anti-oncogenic purposes. METHODS A search of the scientific literature available in the PubMed/Medline was conducted for studies on metabolism and mitochondrial function related to gametogenesis, early development, stem cells and cancers in the reproductive system, notably breast, prostate, ovarian and testicular cancers. RESULTS Both PSCs and some types of cancer cells, particularly reproductive cancers, were found to obtain energy mostly by glycolysis, often reducing mitochondrial activity and oxidative phosphorylation. This strategy links proliferating cells, allowing for the biosynthesis reactions necessary for cell division. Interventions that affect metabolic pathways, and force cells to change their preferences, can lead to shifts in cell status, increasing either pluripotency or differentiation of stem cells, and causing cancer cells to become more or less aggressive. Interestingly metabolic changes in many cases seemed to lead to cell transformation, not necessarily follow it, suggesting a direct role of metabolic choices in influencing the (epi)genetic program of different cell types. CONCLUSIONS There are uncanny similarities between PSCs and cancer cells at the metabolic level. Furthermore, metabolism may also play a direct role in cell status and targeting metabolic pathways could therefore be a promising strategy for both the control of cancer cell proliferation and the regulation of stem cell physiology, in terms of manipulating stem cells toward relevant phenotypes that may be important for tissue engineering, or making cancer cells become less tumorigenic.

Sirtuins in metabolism, stemness and differentiation.

BACKGROUND Pluripotent stem cells promise innovative approaches for enduring diseases, including disease modeling and drug screens. Accordingly, efforts have been undertaken in order to efficiently reprogram somatic cells to pluripotency, and then differentiate them into pure cultures of specific cell lineages. However, the latter step remains mostly elusive, and, in order to better control differentiation and design more efficient differentiation strategies, the cellular mechanisms behind different pluripotency stages that mimic embryonic development are being actively addressed. SCOPE OF REVIEW Metabolism is one of many cellular processes that are in constant adjustment during mammalian embryo development, as well as in pluripotent stem cell establishment and differentiation. Thus, the role of molecular pathways known to be involved in metabolic control has been recently addressed as potential modulators of pluripotency. Notably, mammalian sirtuins have emerged as master regulators of many cellular processes, including epigenetics and metabolism. In this review we address the potential developmental role of sirtuins, with a particular focus on sirtuin 1. MAJOR CONCLUSIONS This review focuses on the most recent studies implying sirtuins as regulators of pluripotency and differentiation of pluripotent stem cells, highlighting metabolic control as associated with the control of pluripotency. It notably stresses the role of sirtuin 1 in these processes, creating parallels between in vitro manipulations and developmental cues. GENERAL SIGNIFICANCE Using metabolic control in order to determine cellular fate, both in terms of somatic cell reprogramming to pluripotency and pluripotent stem cell differentiation, is a topic of increasing interest, and sirtuins are key players in these efforts.

Dichloroacetate, the Pyruvate Dehydrogenase Complex and the Modulation of mESC Pluripotency.

Introduction The pyruvate dehydrogenase (PDH) complex is localized in the mitochondrial matrix catalyzing the irreversible decarboxylation of pyruvate to acetyl-CoA and NADH. For proper complex regulation the E1-α subunit functions as an on/off switch regulated by phosphorylation/dephosphorylation. In different cell types one of the four-pyruvate dehydrogenase kinase isoforms (PDHK1-4) can phosphorylate this subunit leading to PDH inactivation. Our previous results with human Embryonic Stem Cells (hESC), suggested that PDHK could be a key regulator in the metabolic profile of pluripotent cells, as it is upregulated in pluripotent stem cells. Therefore, we wondered if metabolic modulation, via inexpensive pharmacological inhibition of PDHK, could impact metabolism and pluripotency. Methods/Results In order to assess the importance of the PDH cycle in mouse Embryonic Stem Cells (mESC), we incubated cells with the PDHK inhibitor dichloroacetate (DCA) and observed that in its presence ESC started to differentiate. Changes in mitochondrial function and proliferation potential were also found and protein levels for PDH (both phosphorylated and non-phosphorylated) and PDHK1 were monitored. Interestingly, we were also able to describe a possible pathway that involves Hif-1α and p53 during DCA-induced loss of pluripotency. Results with ESCs treated with DCA were comparable to those obtained for cells grown without Leukemia Inhibitor Factor (LIF), used in this case as a positive control for differentiation. Conclusions DCA negatively affects ESC pluripotency by changing cell metabolism and elements related to the PDH cycle, suggesting that PDHK could function as a possible metabolic gatekeeper in ESC, and may be a good target to modulate metabolism and differentiation. Although further molecular biology-based experiments are required, our data suggests that inactive PDH favors pluripotency and that ESC have similar strategies as cancer cells to maintain a glycolytic profile, by using some of the signaling pathways found in the latter cells.

Differentiate or Die: 3-Bromopyruvate and Pluripotency in Mouse Embryonic Stem Cells.

Background Pluripotent embryonic stem cells grown under standard conditions (ESC) have a markedly glycolytic profile, which is shared with many different types of cancer cells. Thus, some therapeutic strategies suggest that pharmacologically shifting cancer cells towards an oxidative phenotype, using glycolysis inhibitors, may reduce cancer aggressiveness. Given the metabolic parallels between cancer and stemness would chemotherapeutical agents have an effect on pluripotency, and could a strategy involving these agents be envisioned to modulate stem cell fate in an accessible manner? In this manuscript we attempted to determine the effects of 3-bromopyruvate (3BrP) in pluripotency. Although it has other intracellular targets, this compound is a potent inhibitor of glycolysis enzymes thought to be important to maintain a glycolytic profile. The goal was also to determine if we could contribute towards a pharmacologically accessible metabolic strategy to influence cell differentiation. Methodology/Principal Findings Mouse embryonic stem cells (mESC) grown under standard pluripotency conditions (in the presence of Leukemia Inducing Factor- LIF) were treated with 3BrP. As a positive control for differentiation other mESCs were grown without LIF. Overall our results demonstrate that 3BrP negatively affects pluripotency, forcing cells to become less glycolytic and with more active mitochondria. These changes in metabolism are correlated with increased differentiation, even under pluripotency conditions (i.e. in the presence of LIF). However, 3BrP also significantly impaired cell function, and may have other roles besides affecting the metabolic profile of mESCs. Conclusions/Findings Treatment of mESCs with 3BrP triggered a metabolic switch and loss of pluripotency, even in the presence of LIF. Interestingly, the positive control for differentiation allowed for a distinction between 3BrP effects and changes associated with spontaneous differentiation/loss of pluripotency in the absence of LIF. Additionally, there was a slight differentiation bias towards mesoderm in the presence of 3BrP. However, the side effects on cellular function suggest that the use of this drug is probably not adequate to efficiently push cells towards specific differentiation fates.

PROneurotrophins and CONSequences

Proneurotrophins were initially thought to be simple inactive precursors, only responsible for promoting the folding of the mature domain and for the regulation of the neurotrophin secretory pathway. However, recent evidence shows that proneurotrophins can be secreted to the extracellular space, bind with high affinity to specific receptor complexes and induce activation of the apoptotic machinery with subsequent cell death of different neuronal populations. These pathways can be activated due to injury and to several neurodegenerative disorders, which promote proneurotrophin secretion to the extracellular space. In addition to neuropathology, extracellular proneurotrophins also play a pivotal role in many other cellular mechanisms in the nervous system. Proneurotrophins were shown to mediate synaptic plasticity, namely long-term depression in hippocampal neurons. They are also important in axonal development, and an increase of pro- to mature neurotrophin ratio has been described as a trigger of cell death. The conversion of proneurotrophins into the respective mature form is controlled by the action of several enzymes and regulators. The failure in this regulation is now considered one of the possible mechanisms responsible for pathological cell death associated to proneurotrophins. Here, we discuss the processes behind proneurotrophin action, with particular focus on proBDNF and proNGF and their regulatory pathways. Additionally, we review the most recent studies concerning proneurotrophin involvement in neuronal death, in several disease-associated states in the CNS and PNS, and discuss future avenues of investigation in the proneurotrophin field.

Pluri-IQ: Quantification of Embryonic Stem Cell Pluripotency through an Image-Based Analysis Software

Summary Image-based assays, such as alkaline phosphatase staining or immunocytochemistry for pluripotent markers, are common methods used in the stem cell field to assess pluripotency. Although an increased number of image-analysis approaches have been described, there is still a lack of software availability to automatically quantify pluripotency in large images after pluripotency staining. To address this need, we developed a robust and rapid image processing software, Pluri-IQ, which allows the automatic evaluation of pluripotency in large low-magnification images. Using mouse embryonic stem cells (mESC) as a model, we combined an automated segmentation algorithm with a supervised machine-learning platform to classify colonies as pluripotent, mixed, or differentiated. In addition, Pluri-IQ allows the automatic comparison between different culture conditions. This efficient user-friendly open-source software can be easily implemented in images derived from pluripotent cells or cells that express pluripotent markers (e.g., OCT4-GFP) and can be routinely used, decreasing image assessment bias.

Metabolic and Mechanical Cues Regulating Pluripotent Stem Cell Fate

The ability to shift between metabolic states and to tightly regulate cellular mechanical properties have been described as crucial events in the achievement of correct embryonic development. Indeed, metabolic and mechanical manipulations in vitro have led to the discovery of new methods to control cell fate. As these two modulators are usually studied separately, in this review article, we describe how cellular mechanics and metabolic characteristics regulate embryonic development in vivo and describe the role of these cues in the regulation of pluripotency and differentiation in vitro. We also pinpoint possible connections between metabolism and mechanotransduction, highlighting recent findings in the Yes-associated protein, phosphoinositide 3-kinase, and AMP-activated protein kinase signaling pathways, and how they may be relevant in modulating cell fate in other contexts.

Erratum: Pluri-IQ: Quantification of Embryonic Stem Cell Pluripotency through an Image-Based Analysis Software (Stem Cell Reports (2017) 9(2) (697–709) (S2213671117302692) (10.1016/j.stemcr.2017.06.006))

(Stem Cell Reports 9, 697–709; August 8, 2017) In the originally published version of this Resource, an error appeared in Equation 4 in the Experimental Procedures. The error does not affect the conclusions of the paper; all of the calculations with the Dice index were done with the correct formula. Equation 4 has been corrected in the paper online, and appears correctly below. The authors apologize for any confusion this mistake may have caused.

Data on the potential impact of food supplements on the growth of mouse embryonic stem cells

The use of new compounds as dietary supplements is increasing, but little is known in terms of possible consequences of their use. Pluripotent stem cells are a promising research tool for citotoxicological research for evaluation of proliferation, cell death, pluripotency and differentiation. Using the mouse embryonic stem cell (mESC) model, we present data on three different compounds that have been proposed as new potential supplements for co-adjuvant disease treatments: kaempferol, berberine and Tauroursodeoxycholic acid (TUDCA). Cell number and viability were monitored following treatment with increased concentrations of each drug in pluripotent culture conditions.