Folmes, Clifford D.L.

ABSTRACT The rare human neuroendocrine tumors pheochromocytoma and paraganglioma (PPGL) can result from loss of mitochondrial succinate dehydrogenase. The resulting succinate accumulation is tumorigenic in certain neuroendocrine cells. Here, we explore two theoretical approaches to mitigate tumorigenic succinate accumulation in a cell culture model of PPGL. We first study a gene replacement strategy using transposition technology, and conclude that many of the changes in mitochondrial morphology, oxidative cell metabolism and succinate accumulation can be reversed by this process. We then investigate whether riboflavin supplementation has the potential to rescue succinate dehydrogenase activity in the intact SDHA catalytic subunit to suppress succinate accumulation even in the absence of SDHB. We show that this latter strategy is not successful.

Growing evidence indicates that metabolites and energy metabolism play an active rather than consequential role in regulating cellular fate. Cardiac development requires dramatic metabolic remodeling from relying primarily on glycolysis in pluripotent stem cells (PSCs) to oxidizing a wide array of energy substrates to match the high bioenergetic demands of continuous contraction in the developed heart. However, a detailed analysis of how remodeling of energy metabolism contributes to human cardiac development is lacking. Using dynamic multiple reaction monitoring metabolomics of central carbon metabolism, we evaluated temporal changes in energy metabolism during human PSC 3D cardiac lineage specification. Significant metabolic remodeling occurs during the complete differentiation, yet temporal analysis revealed that most changes occur during transitions from pluripotency to mesoderm (day 1) and mesoderm to early cardiac (day 5), with limited maturation of cardiac metabolism beyond day 5. Real-time metabolic analysis demonstrated that while hPSC cardiomyocytes (hPSC-CM) showed elevated rates of oxidative metabolism compared to PSCs, they still retained high glycolytic rates, confirming an immature metabolic phenotype. These observations support the opportunity to metabolically optimize the differentiation process to support lineage specification and maturation of hPSC-CMs.

Abstract We have previously shown that a frequent loss of endosulfatase HSulf-1 in ovarian cancer enhances the heparin binding growth factor signaling which subsequently induces and potentiates tumorigenesis. Our recent data indicate that knockdown of HSulf-1 in ovarian cancer cells significantly alter levels of several metabolites which is comparable to the deranged metabolite profiles in primary ovarian tumors. Here we report that the metabolites associated with lipid metabolism are altered in such a way that the cells start manifesting a “lipogenic phenotype” characterized by an increased fatty acid synthesis, storage and mitochondrial beta oxidation. The lipogenic phenotype is associated with an accumulation of cytoplasmic lipid droplets (LDs) in a stably HSulf-1 knockdown cells [OV202 (Sh1/2) and OV2223 cells]. Presence of LDs is evidenced by BODIPY staining and transmission electron microscopy (TEM) analysis in Sh1/2 and a non-transduced control (NTC) cells. Conversely, enhanced expression of HSulf-1 in TOV21G, SKOV3, and C13 cells also resulted in reducing the number of LDs. More importantly, MEFs (mouse embryonic fibroblasts) from HSulf-1 knockout mice also showed an increased LD biogenesis compared to the wild type MEFs. Microarray analysis identified cytoplasmic phospholipase A2 (cPLA2; an enzyme involved in LD biogenesis) as one of the genes that was significantly upregulated in OV202 Sh1 and Sh2 cells. We found that Sh1 and Sh2 cells show an increased phosphorylation (ser505) of cytoplasmic phospholipase A2 (p-cPLA2) compared to NTC cells. Phosphorylation at ser505 is vital for the enzyme activity of cPLA2 and also necessary for LD biogenesis. We inhibited the LD biogenesis in Sh1 cells by a pharmacological inhibitor of cPLA2 (AACOCF3 and MAFP) as well by shRNA mediated knockdown of endogenous cPLA2. Further, we stimulated NTC and Sh1 cells with heparin binding growth factor (50ng/ml) for different time points and found an elevated phosphorylation of cPLA2 and extracellular signal related kinase (ERK), which is also inhibited by a pre-treatment of cells with cPLA2 inhibitor. Interestingly, inhibition of phospho-ERK by U0126 (MEK inhibitor) also inhibits the phosphorylation of cPLA2. These findings strongly suggest that loss of HSulf-1 promotes lipid metabolism in the ovarian cancer cells via activation of cPLA2 via an ERK dependent pathway. The present work highlights a novel role HSulf-1 in regulating lipid metabolism which could lead to a better understanding of tumorigenesis and identification of new targets for tumor therapy. Citation Format: Debarshi Roy, Xiaoping He, Ashwani Khurana, Susmita Mondal, Deok-Beom Jung, Sung Kim, Thomas Dierks, Clifford Folmes, Andre Terzic, Viji Shridhar. HSulf-1 loss in ovarian cancer cells induces lipid droplet biogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2439. doi:10.1158/1538-7445.AM2014-2439

Decoding stem cell metabolism has implicated a tight linkage between energy metabolism and cell fate regulation, a dynamic interplay vital in the execution of developmental and differentiation programs. The inherent plasticity in energy metabolism enables prioritisation of metabolic pathways in support of stage-specific demands. Beyond traditional support of energetic needs, intermediate metabolism may also dictate cell fate choices through regulation of cellular signalling and epigenetic regulation of gene expression. The notion of a ‘metabolism-centric’ control of stem cell differentiation has been informed by developmental embryogenesis based upon an on-demand paradigm paramount in defining diverse developmental behaviours, from a post-fertilisation nascent zygote to complex organogenesis leading to adequate tissue formation and maturation. Monitored through natural or bioengineered stem cell surrogates, nutrient-responsive metabolites are identified as mediators of cross-talk between metabolic flux, cell signalling and epigenetic regulation charting, collectively, whether a cell will self-renew to maintain progenitor pools, lineage specify to ensure tissue (re)generation or remain quiescent to curb stress damage. Thus, bioenergetics are increasingly recognised as integral in governing stemness and associated organogenic decisions, paving the way for metabolism-defined targets in control of embryology, stem cell biology and tissue regeneration.

Significance: Metabolism-dependent generation of reactive oxygen species (ROS) and associated oxidative damage have been traditionally linked to impaired homeostasis and cellular death. Beyond the adverse effects of ROS accumulation, increasing evidence implicates redox status as a regulator of vital cellular processes. Recent Advances: Emerging studies on the molecular mechanisms guiding stem cell fate decisions indicate a role for energy metabolism in regulating the fundamental ability of maintaining stemness versus undergoing lineage-specific differentiation. Stem cells have evolved protective metabolic phenotypes to minimize reactive oxygen generation through oxidative metabolism and support antioxidant scavenging through glycolysis and the pentose phosphate pathway. Critical Issues: While the dynamics in ROS generation has been correlated with stem cell function, the intimate mechanisms by which energy metabolism regulates ROS to impact cellular fate remain to be deciphered. Future Directions: Decoding the linkage between nutrient sensing, energy metabolism, and ROS in regulating cell fate decisions would offer a redox-dependent strategy to regulate stemness and lineage specification. Antioxid. Redox Signal . 21, 1648–1659.

Abstract Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.

Abstract Pluripotent stem cells have been the focus of bioengineering efforts designed to generate regenerative products, yet harnessing therapeutic capacity while minimizing risk of dysregulated growth remains a challenge. The risk of residual undifferentiated stem cells within a differentiated progenitor population requires a targeted approach to eliminate contaminating cells prior to delivery. In this study we aimed to validate a toxicity strategy that could selectively purge pluripotent stem cells in response to DNA damage and avoid risk of uncontrolled cell growth upon transplantation. Compared with somatic cell types, embryonic stem cells and induced pluripotent stem cells displayed hypersensitivity to apoptotic induction by genotoxic agents. Notably, hypersensitivity in pluripotent stem cells was stage-specific and consistently lost upon in vitro differentiation, with the mean half-maximal inhibitory concentration increasing nearly 2 orders of magnitude with tissue specification. Quantitative polymerase chain reaction and Western blotting demonstrated that the innate response was mediated through upregulation of the BH3-only protein Puma in both natural and induced pluripotent stem cells. Pretreatment with genotoxic etoposide purged hypersensitive pluripotent stem cells to yield a progenitor population refractory to teratoma formation upon transplantation. Collectively, this study exploits a hypersensitive apoptotic response to DNA damage within pluripotent stem cells to decrease risk of dysregulated growth and augment the safety profile of transplant-ready, bioengineered progenitor cells.