Publications by authors named "Roger Sik Yin Foo"

24 Publications

  • Page 1 of 1

Regulatory enhancer profiling of mesenchymal-type gastric cancer reveals subtype-specific epigenomic landscapes and targetable vulnerabilities.

Gut 2022 Jul 11. Epub 2022 Jul 11.

Genome Institute of Singapore, Agency for Science, Technology and Research, Singapore

Objective: Gastric cancer (GC) comprises multiple molecular subtypes. Recent studies have highlighted mesenchymal-subtype GC (Mes-GC) as a clinically aggressive subtype with few treatment options. Combining multiple studies, we derived and applied a consensus Mes-GC classifier to define the Mes-GC enhancer landscape revealing disease vulnerabilities.

Design: Transcriptomic profiles of ~1000 primary GCs and cell lines were analysed to derive a consensus Mes-GC classifier. Clinical and genomic associations were performed across >1200 patients with GC. Genome-wide epigenomic profiles (H3K27ac, H3K4me1 and assay for transposase-accessible chromatin with sequencing (ATAC-seq)) of 49 primary GCs and GC cell lines were generated to identify Mes-GC-specific enhancer landscapes. Upstream regulators and downstream targets of Mes-GC enhancers were interrogated using chromatin immunoprecipitation followed by sequencing (ChIP-seq), RNA sequencing, CRISPR/Cas9 editing, functional assays and pharmacological inhibition.

Results: We identified and validated a 993-gene cancer-cell intrinsic Mes-GC classifier applicable to retrospective cohorts or prospective single samples. Multicohort analysis of Mes-GCs confirmed associations with poor patient survival, therapy resistance and few targetable genomic alterations. Analysis of enhancer profiles revealed a distinctive Mes-GC epigenomic landscape, with as a master regulator of Mes-GC enhancers and Mes-GCs exhibiting preferential sensitivity to TEAD1 pharmacological inhibition. Analysis of Mes-GC super-enhancers also highlighted kinase as a downstream target, with synergistic effects observed between NUAK1 inhibition and cisplatin treatment.

Conclusion: Our results establish a consensus Mes-GC classifier applicable to multiple transcriptomic scenarios. Mes-GCs exhibit a distinct epigenomic landscape, and TEAD1 inhibition and combinatorial NUAK1 inhibition/cisplatin may represent potential targetable options.
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http://dx.doi.org/10.1136/gutjnl-2021-326483DOI Listing
July 2022

11C-Metomidate PET-CT versus adrenal vein sampling to subtype primary aldosteronism: a prospective clinical trial.

J Hypertens 2022 Jun;40(6):1179-1188

Yong Loo Lin School of Medicine, National University of Singapore (NUS).

Objective: Adrenal vein sampling (AVS) is recommended to subtype primary aldosteronism, but it is technically challenging. We compared 11C-Metomidate-PET-computed tomography (PET-CT) and AVS for subtyping of primary aldosteronism.

Methods: Patients with confirmed primary aldosteronism underwent both AVS and 11C-Metomidate PET-CT (post-dexamethasone). All results were reviewed at a multidisciplinary meeting to decide on final subtype diagnosis. Primary outcome was accuracy of PET versus AVS to diagnosis of unilateral primary aldosteronism based on post-surgical biochemical cure. Secondary outcome was accuracy of both tests to final subtype diagnosis.

Results: All 25 patients recruited underwent PET and successful AVS (100%). Final diagnosis was unilateral in 22 patients, bilateral in two and indeterminate in one due to discordant lateralization. Twenty patients with unilateral primary aldosteronism underwent surgery, with 100% complete biochemical success, and 75% complete/partial clinical success. For the primary outcome, sensitivity of PET was 80% [95% confidence interval (95% CI): 56.3-94.3] and AVS was 75% (95% CI: 50.9-91.3). For the secondary outcome, sensitivity and specificity of PET was 81.9% (95% CI: 59.7-94.8) and 100% (95% CI: 15.8-100), and AVS was 68.2% (95% CI: 45.1-86.1) and 100% (95% CI: 15.8-100), respectively. Twelve out of 20 (60%) patients had both PET and AVS lateralization, four (20%) PET-only, three (15%) AVS-only, while one patient did not lateralize on PET or AVS. Post-surgery outcomes did not differ between patients identified by either test.

Conclusion: In our pilot study, 11C-Metomidate PET-CT performed comparably to AVS, and this should be validated in larger studies. PET identified patients with unilateral primary aldosteronism missed on AVS, and these tests could be used together to identify more patients with unilateral primary aldosteronism.

Video Abstract: http://links.lww.com/HJH/B918.
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http://dx.doi.org/10.1097/HJH.0000000000003132DOI Listing
June 2022

Time-restricted feeding modulates the DNA methylation landscape, attenuates hallmark neuropathology and cognitive impairment in a mouse model of vascular dementia.

Theranostics 2022 21;12(7):3007-3023. Epub 2022 Mar 21.

School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.

Vascular dementia (VaD) is the second most common cause of dementia worldwide. The increasing contribution of lifestyle-associated risk factors to VaD has pointed towards gene-environment interactions (i.e. epigenetics). This study thus aims to investigate the DNA methylation landscape in a chronic cerebral hypoperfusion (CCH) mouse model of VaD. As a nexus between the gene-environment interaction, intermittent fasting (IF) was introduced as a prophylactic intervention. Bilateral common carotid artery stenosis (BCAS) was used to induce CCH by placing micro-coils of 0.18 mm in each common carotid artery of the mice. The coils were left in the mice for 7, 15 and 30 days to study temporal differences. IF was introduced for 16 h daily for 4 months prior to BCAS. Reduced Representation Bisulfite Sequencing (RRBS) was used to study the DNA methylation landscape. Cognitive impairment was measured using Barnes Maze Test. White matter lesions (WML) and neuronal loss were measured using Luxol fast blue staining and cresyl violet staining respectively. IF mice subjected to CCH displayed significantly better cognitive learning ability and memory, improved neuropathological alterations with reduced WMLs and neuronal loss. Modulation of DNA methylation patterns in the cortex of AL CCH mice was re-modelled and signs of reversal was observed in IF CCH mice across all three timepoints. These findings provide an understanding of how IF may protect the brain against damage caused by CCH and show promise in offering potential beneficial effects in mitigating the neuropathology and cognitive deficits in VaD.
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http://dx.doi.org/10.7150/thno.71815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9065201PMC
March 2022

8-Oxoguanine DNA Glycosylase (OGG1) Deficiency Exacerbates Doxorubicin-Induced Cardiac Dysfunction.

Oxid Med Cell Longev 2022 27;2022:9180267. Epub 2022 Mar 27.

Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Japan.

Doxorubicin is an anthracycline widely used for the treatment of various cancers; however, the drug has a common deleterious side effect, namely a dose-dependent cardiotoxicity. Doxorubicin treatment increases the generation of reactive oxygen species, which leads to oxidative stress in the cardiac cells and ultimately DNA damage and cell death. The most common DNA lesion produced by oxidative stress is 7,8-dihydro-8-oxoguanine (8-oxoguanine), and the enzyme responsible for its repair is the 8-oxoguanine DNA glycosylase (OGG1), a base excision repair enzyme. Here, we show that the OGG1 deficiency has no major effect on cardiac function at baseline or with pressure overload; however, we found an exacerbation of cardiac dysfunction as well as a higher mortality in knockout mice treated with doxorubicin. Our transcriptomic analysis also showed a more extensive dysregulation of genes in the hearts of knockout mice with an enrichment of genes involved in inflammation. These results demonstrate that OGG1 attenuates doxorubicin-induced cardiotoxicity and thus plays a role in modulating drug-induced cardiomyopathy.
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http://dx.doi.org/10.1155/2022/9180267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8981022PMC
April 2022

Dynamic phenotypic heterogeneity and the evolution of multiple RNA subtypes in hepatocellular carcinoma: the PLANET study.

Natl Sci Rev 2022 Mar 29;9(3):nwab192. Epub 2021 Oct 29.

Department of Pathology, Singapore General Hospital, Singapore 169608, Singapore.

Intra-tumor heterogeneity (ITH) is a key challenge in cancer treatment, but previous studies have focused mainly on the genomic alterations without exploring phenotypic (transcriptomic and immune) heterogeneity. Using one of the largest prospective surgical cohorts for hepatocellular carcinoma (HCC) with multi-region sampling, we sequenced whole genomes and paired transcriptomes from 67 HCC patients (331 samples). We found that while genomic ITH was rather constant across stages, phenotypic ITH had a very different trajectory and quickly diversified in stage II patients. Most strikingly, 30% of patients were found to contain more than one transcriptomic subtype within a single tumor. Such phenotypic ITH was found to be much more informative in predicting patient survival than genomic ITH and explains the poor efficacy of single-target systemic therapies in HCC. Taken together, we not only revealed an unprecedentedly dynamic landscape of phenotypic heterogeneity in HCC, but also highlighted the importance of studying phenotypic evolution across cancer types.
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http://dx.doi.org/10.1093/nsr/nwab192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8973408PMC
March 2022

Integrative epigenomic and transcriptomic analyses reveal metabolic switching by intermittent fasting in brain.

Geroscience 2022 Mar 31. Epub 2022 Mar 31.

Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

Intermittent fasting (IF) remains the most effective intervention to achieve robust anti-aging effects and attenuation of age-related diseases in various species. Epigenetic modifications mediate the biological effects of several environmental factors on gene expression; however, no information is available on the effects of IF on the epigenome. Here, we first found that IF for 3 months caused modulation of H3K9 trimethylation (H3K9me) in the cerebellum, which in turn orchestrated a plethora of transcriptomic changes involved in robust metabolic switching processes commonly observed during IF. Second, a portion of both the epigenomic and transcriptomic modulations induced by IF was remarkably preserved for at least 3 months post-IF refeeding, indicating that memory of IF-induced epigenetic changes was maintained. Notably, though, we found that termination of IF resulted in a loss of H3K9me regulation of the transcriptome. Collectively, our study characterizes the novel effects of IF on the epigenetic-transcriptomic axis, which controls myriad metabolic processes. The comprehensive analyses undertaken in this study reveal a molecular framework for understanding how IF impacts the metabolo-epigenetic axis of the brain and will serve as a valuable resource for future research.
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http://dx.doi.org/10.1007/s11357-022-00537-zDOI Listing
March 2022

Integrative epigenomic and high-throughput functional enhancer profiling reveals determinants of enhancer heterogeneity in gastric cancer.

Genome Med 2021 10 11;13(1):158. Epub 2021 Oct 11.

Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore, 169857, Singapore.

Background: Enhancers are distal cis-regulatory elements required for cell-specific gene expression and cell fate determination. In cancer, enhancer variation has been proposed as a major cause of inter-patient heterogeneity-however, most predicted enhancer regions remain to be functionally tested.

Methods: We analyzed 132 epigenomic histone modification profiles of 18 primary gastric cancer (GC) samples, 18 normal gastric tissues, and 28 GC cell lines using Nano-ChIP-seq technology. We applied Capture-based Self-Transcribing Active Regulatory Region sequencing (CapSTARR-seq) to assess functional enhancer activity. An Activity-by-contact (ABC) model was employed to explore the effects of histone acetylation and CapSTARR-seq levels on enhancer-promoter interactions.

Results: We report a comprehensive catalog of 75,730 recurrent predicted enhancers, the majority of which are GC-associated in vivo (> 50,000) and associated with lower somatic mutation rates inferred by whole-genome sequencing. Applying CapSTARR-seq to the enhancer catalog, we observed significant correlations between CapSTARR-seq functional activity and H3K27ac/H3K4me1 levels. Super-enhancer regions exhibited increased CapSTARR-seq signals compared to regular enhancers, even when decoupled from native chromatin contexture. We show that combining histone modification and CapSTARR-seq functional enhancer data improves the prediction of enhancer-promoter interactions and pinpointing of germline single nucleotide polymorphisms (SNPs), somatic copy number alterations (SCNAs), and trans-acting TFs involved in GC expression. We identified cancer-relevant genes (ING1, ARL4C) whose expression between patients is influenced by enhancer differences in genomic copy number and germline SNPs, and HNF4α as a master trans-acting factor associated with GC enhancer heterogeneity.

Conclusions: Our results indicate that combining histone modification and functional assay data may provide a more accurate metric to assess enhancer activity than either platform individually, providing insights into the relative contribution of genetic (cis) and regulatory (trans) mechanisms to GC enhancer functional heterogeneity.
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http://dx.doi.org/10.1186/s13073-021-00970-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8504099PMC
October 2021

Germline genome modification through novel political, ethical, and social lenses.

PLoS Genet 2021 09 9;17(9):e1009741. Epub 2021 Sep 9.

School of Social Sciences, Cardiff University, Cardiff, United Kingdom.

Much has been written about gene modifying technologies (GMTs), with a particularly strong focus on human germline genome editing (HGGE) sparked by its unprecedented clinical research application in 2018, shocking the scientific community. This paper applies political, ethical, and social lenses to aspects of HGGE to uncover previously underexplored considerations that are important to reflect on in global discussions. By exploring 4 areas-(1) just distribution of HGGE benefits through a realist lens; (2) HGGE through a national interest lens; (3) "broad societal consensus" through a structural injustice lens; and (4) HGGE through a scientific trustworthiness lens-a broader perspective is offered, which ultimately aims to enrich further debates and inform well-considered solutions for developments in this field. The application of these lenses also brings to light the fact that all discussions about scientific developments involve a conscious or unconscious application of a lens that shapes the direction of our thinking.
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http://dx.doi.org/10.1371/journal.pgen.1009741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428543PMC
September 2021

Disrupting the LINC complex by AAV mediated gene transduction prevents progression of Lamin induced cardiomyopathy.

Nat Commun 2021 08 5;12(1):4722. Epub 2021 Aug 5.

Skin Research Institute (SRIS), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

Mutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3-4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1's function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.
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http://dx.doi.org/10.1038/s41467-021-24849-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8342462PMC
August 2021

Dimethyl sulfoxide (DMSO) enhances direct cardiac reprogramming by inhibiting the bromodomain of coactivators CBP/p300.

J Mol Cell Cardiol 2021 11 17;160:15-26. Epub 2021 Jun 17.

Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive, Singapore 117599, Singapore; Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore. Electronic address:

Aims: Direct cardiac reprogramming represents an attractive way to reversing heart damage caused by myocardial infarction because it removes fibroblasts, while also generating new functional cardiomyocytes. Yet, the main hurdle for bringing this technique to the clinic is the lack of efficacy with current reprogramming protocols. Here, we describe our unexpected discovery that DMSO is capable of significantly augmenting direct cardiac reprogramming in vitro.

Methods And Results: Upon induction with cardiac transcription factors- Gata4, Hand2, Mef2c and Tbx5 (GHMT), the treatment of mouse embryonic fibroblasts (MEFs) with 1% DMSO induced ~5 fold increase in Myh6-mCherry+ cells, and significantly upregulated global expression of cardiac genes, including Myh6, Ttn, Nppa, Myh7 and Ryr2. RNA-seq confirmed upregulation of cardiac gene programmes and downregulation of extracellular matrix-related genes. Treatment of TGF-β1, DMSO, or SB431542, and the combination thereof, revealed that DMSO most likely targets a separate but parallel pathway other than TGF-β signalling. Subsequent experiments using small molecule screening revealed that DMSO enhances direct cardiac reprogramming through inhibition of the CBP/p300 bromodomain, and not its acetyltransferase property.

Conclusion: In conclusion, our work points to a direct molecular target of DMSO, which can be used for augmenting GHMT-induced direct cardiac reprogramming and possibly other cell fate conversion processes.
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http://dx.doi.org/10.1016/j.yjmcc.2021.06.008DOI Listing
November 2021

Effects of extended pharmacological disruption of zebrafish embryonic heart biomechanical environment on cardiac function, morphology, and gene expression.

Dev Dyn 2021 12 4;250(12):1759-1777. Epub 2021 Jun 4.

Department of Biomedical Engineering, National University of Singapore, Singapore, Singapore.

Background: Biomechanical stimuli are known to be important to cardiac development, but the mechanisms are not fully understood. Here, we pharmacologically disrupted the biomechanical environment of wild-type zebrafish embryonic hearts for an extended duration and investigated the consequent effects on cardiac function, morphological development, and gene expression.

Results: Myocardial contractility was significantly diminished or abolished in zebrafish embryonic hearts treated for 72 hours from 2 dpf with 2,3-butanedione monoxime (BDM). Image-based flow simulations showed that flow wall shear stresses were abolished or significantly reduced with high oscillatory shear indices. At 5 dpf, after removal of BDM, treated embryonic hearts were maldeveloped, having disrupted cardiac looping, smaller ventricles, and poor cardiac function (lower ejected flow, bulboventricular regurgitation, lower contractility, and slower heart rate). RNA sequencing of cardiomyocytes of treated hearts revealed 922 significantly up-regulated genes and 1,698 significantly down-regulated genes. RNA analysis and subsequent qPCR and histology validation suggested that biomechanical disruption led to an up-regulation of inflammatory and apoptotic genes and down-regulation of ECM remodeling and ECM-receptor interaction genes. Biomechanics disruption also prevented the formation of ventricular trabeculation along with notch1 and erbb4a down-regulation.

Conclusions: Extended disruption of biomechanical stimuli caused maldevelopment, and potential genes responsible for this are identified.
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http://dx.doi.org/10.1002/dvdy.378DOI Listing
December 2021

Enhancing the cardiovascular protective effects of a healthy dietary pattern with wolfberry (Lycium barbarum): A randomized controlled trial.

Am J Clin Nutr 2021 07;114(1):80-89

Department of Food Science & Technology, Faculty of Science, National University of Singapore, Singapore, Singapore.

Background: The consumption of wolfberry (Lycium barbarum), a rich source of carotenoids and bioactive polysaccharides, may serve as a potential dietary strategy for cardiovascular disease (CVD) risk management although limited studies examined its effects as whole fruits.

Objectives: To investigate the impact of wolfberry consumption as part of a healthy dietary pattern on vascular health-related outcomes and classical CVD risk factors in middle-aged and older adults in Singapore.

Methods: This is a 16-week, parallel design, randomized controlled trial. All participants (n = 40) received dietary counselling to follow healthy dietary pattern recommendations with the wolfberry group given additional instructions to cook and consume 15 g/d whole, dried wolfberry with their main meals. Biomarkers of vascular function (flow-mediated dilation, plasma total nitrate/nitrite, endothelin-1, and intercellular adhesion molecule-1), vascular structure (carotid intima-media thickness) and vascular regeneration (endothelial progenitor cell count, plasma angiopoietin 1 and angiopoietin 2), were assessed at baseline and postintervention. Serum lipid-lipoproteins and blood pressure were evaluated every 4 weeks.

Results: All participants showed an improved compliance toward the healthy dietary pattern. This was coupled with marked rises in total nitrate/nitrite concentrations (mean change wolfberry: 3.92 ± 1.73 nmol/mL; control: 5.01 ± 2.55 nmol/L) and reductions in endothelin-1 concentrations (wolfberry: -0.19 ± 0.06 pg/mL; control: -0.15 ± 0.08 pg/mL). Compared with the control which depicted no changes from baseline, the wolfberry group had a significantly higher HDL cholesterol (0.08 ± 0.04 mmol/L), as well as lower Framingham predicted long-term CVD risk (-0.8 ± 0.5%) and vascular age (-1.9 ± 1.0 y) postintervention. No differences were observed in the other vascular health-related outcomes.

Conclusions: In middle-aged and older adults, adherence to a healthy dietary pattern improves vascular tone. Incorporating wolfberry to the diet further improves blood lipid-lipoprotein profile and may lower long-term CVD risk. This study was registered at clinicatrials.gov as NCT03535844.
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http://dx.doi.org/10.1093/ajcn/nqab062DOI Listing
July 2021

Cohort profile: the Diet and Healthy Aging (DaHA) study in Singapore.

Aging (Albany NY) 2020 11 18;12(23):23889-23899. Epub 2020 Nov 18.

Department of Psychological Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

How diet is related with cognition and health has not been systematically examined in Asians whose eating habits are very different from their counterparts in the West and the biological mechanisms underlying such links are not well known yet. The diet and healthy aging (DaHA) study is a community-based longitudinal study conducted to examine the role of diet and nutrition in promoting cognitive, emotional, and physical health among community-living elderly Singaporeans. The first wave of DaHA, conducted from 2011 to 2017, provided detailed information on diet and baseline cognitive function and health from 1010 community-living elderly in Singapore. Biomarkers of oxidative stress, systemic inflammation, and genetic information were collected. The ongoing second wave of DaHA is conducted from 2017 to 2020, which provides follow- up assessments using established cognitive tests and clinical tools. This well-characterized cohort, with its archived biological samples and high-quality data on diet and lifestyle factors will allow researchers to explore the relationships among diet, nutrition, genes, cognition, mental and physical health in an extremely cost-effective manner. Translations of the research findings into clinical and public health practices will potentially help to promote cognitive health at the population level and reduce healthcare costs related to cognitive impairment.
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http://dx.doi.org/10.18632/aging.104051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762480PMC
November 2020

International Reporting Mechanism for Unethical Germline Gene Editing Experiments Is Needed.

Trends Biotechnol 2021 05 4;39(5):427-430. Epub 2020 Nov 4.

Science, Health, and Policy-Relevant Ethics in Singapore (SHAPES), Centre for Biomedical Ethics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

Recent reporting found that a number of scientists internationally knew about the experiment resulting in the birth of the first gene-edited babies well before the news broke. Because scientists have a responsibility to reveal such activities, an international governance mechanism for reporting unethical gene editing experiments should be established.
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http://dx.doi.org/10.1016/j.tibtech.2020.10.001DOI Listing
May 2021

Mitochondrial Substrate Utilization Regulates Cardiomyocyte Cell Cycle Progression.

Nat Metab 2020 02 20;2(2):167-178. Epub 2020 Feb 20.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

The neonatal mammalian heart is capable of regeneration for a brief window of time after birth. However, this regenerative capacity is lost within the first week of life, which coincides with a postnatal shift from anaerobic glycolysis to mitochondrial oxidative phosphorylation, particularly towards fatty-acid utilization. Despite the energy advantage of fatty-acid beta-oxidation, cardiac mitochondria produce elevated rates of reactive oxygen species when utilizing fatty acids, which is thought to play a role in cardiomyocyte cell-cycle arrest through induction of DNA damage and activation of DNA-damage response (DDR) pathway. Here we show that inhibiting fatty-acid utilization promotes cardiomyocyte proliferation in the postnatatal heart. First, neonatal mice fed fatty-acid deficient milk showed prolongation of the postnatal cardiomyocyte proliferative window, however cell cycle arrest eventually ensued. Next, we generated a tamoxifen-inducible cardiomyocyte-specific, pyruvate dehydrogenase kinase 4 (PDK4) knockout mouse model to selectively enhance oxidation of glycolytically derived pyruvate in cardiomyocytes. Conditional PDK4 deletion resulted in an increase in pyruvate dehydrogenase activity and consequently an increase in glucose relative to fatty-acid oxidation. Loss of PDK4 also resulted in decreased cardiomyocyte size, decreased DNA damage and expression of DDR markers and an increase in cardiomyocyte proliferation. Following myocardial infarction, inducible deletion of PDK4 improved left ventricular function and decreased remodelling. Collectively, inhibition of fatty-acid utilization in cardiomyocytes promotes proliferation, and may be a viable target for cardiac regenerative therapies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331943PMC
February 2020

Extracellular vesicles from human embryonic stem cell-derived cardiovascular progenitor cells promote cardiac infarct healing through reducing cardiomyocyte death and promoting angiogenesis.

Cell Death Dis 2020 05 11;11(5):354. Epub 2020 May 11.

CAS Key Laboratory of Tissue Microenvironment and Tumor, Laboratory of Molecular Cardiology, Shanghai Institute of Nutrition and Health, University of Chinese Academy of Sciences (CAS), CAS, Shanghai, 200031, P. R. China.

Human pluripotent stem cells (hPSCs)-derived cardiovascular progenitor cells (CVPCs) are a promising source for myocardial repair, while the mechanisms remain largely unknown. Extracellular vesicles (EVs) are known to mediate cell-cell communication, however, the efficacy and mechanisms of hPSC-CVPC-secreted EVs (hCVPC-EVs) in the infarct healing when given at the acute phase of myocardial infarction (MI) are unknown. Here, we report the cardioprotective effects of the EVs secreted from hESC-CVPCs under normoxic (EV-N) and hypoxic (EV-H) conditions in the infarcted heart and the long noncoding RNA (lncRNA)-related mechanisms. The hCVPC-EVs were confirmed by electron microscopy, nanoparticle tracking, and immunoblotting analysis. Injection of hCVPC-EVs into acutely infracted murine myocardium significantly improved cardiac function and reduced fibrosis at day 28 post MI, accompanied with the improved vascularization and cardiomyocyte survival at border zones. Consistently, hCVPC-EVs enhanced the tube formation and migration of human umbilical vein endothelial cells (HUVECs), improved the cell viability, and attenuated the lactate dehydrogenase release of neonatal rat cardiomyocytes (NRCMs) with oxygen glucose deprivation (OGD) injury. Moreover, the improvement of the EV-H in cardiomyocyte survival and tube formation of HUVECs was significantly better than these in the EV-N. RNA-seq analysis revealed a high abundance of the lncRNA MALAT1 in the EV-H. Its abundance was upregulated in the infarcted myocardium and cardiomyocytes treated with hCVPC-EVs. Overexpression of human MALAT1 improved the cell viability of NRCM with OGD injury, while knockdown of MALAT1 inhibited the hCVPC-EV-promoted tube formation of HUVECs. Furthermore, luciferase activity assay, RNA pull-down, and manipulation of miR-497 levels showed that MALAT1 improved NRCMs survival and HUVEC tube formation through targeting miR-497. These results reveal that hCVPC-EVs promote the infarct healing through improvement of cardiomyocyte survival and angiogenesis. The cardioprotective effects of hCVPC-EVs can be enhanced by hypoxia-conditioning of hCVPCs and are partially contributed by MALAT1 via targeting the miRNA.
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http://dx.doi.org/10.1038/s41419-020-2508-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214429PMC
May 2020

Genomic and epigenomic EBF1 alterations modulate TERT expression in gastric cancer.

J Clin Invest 2020 06;130(6):3005-3020

Cancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore.

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.
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http://dx.doi.org/10.1172/JCI126726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260007PMC
June 2020

Circles in the heart and cardiovascular system.

Cardiovasc Res 2020 02;116(2):269-278

Cardiovascular Research Institute, National University Health Systems, MD6 Centre for Translational Medicine, 14 Medical Drive, Singapore 117599, Singapore.

The combination of next-generation sequencing, advanced bioinformatics analysis, and molecular research has now established circular RNAs (circRNAs) as a heterogeneous group of non-coding RNA that is widely and abundantly expressed. CircRNAs are single-stranded RNA, covalently backspliced to form closed circular loops. Different models of back-splicing have been proposed, and mechanisms for circRNA function include sequestering microRNAs, direct interaction with proteins, regulation of transcription, and translation. Exploring the role of circRNAs in different disease settings, and understanding how they contribute to disease progression promises to provide valuable insight into potential novel therapeutic approaches. Here, we review the growing number of published research on circRNAs in the heart and cardiovascular system and summarize the circRNAs that have been implicated in disease.
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http://dx.doi.org/10.1093/cvr/cvz227DOI Listing
February 2020

Dissecting Chromatin Architecture for Novel Cardiovascular Disease Targets.

Circulation 2019 08 5;140(6):446-448. Epub 2019 Aug 5.

Departments of Anesthesiology, Medicine and Physiology, David Geffen School of Medicine at UCLA (M.R.-G., T.M.V.).

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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.039287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989048PMC
August 2019

Targeting the highly abundant circular RNA circSlc8a1 in cardiomyocytes attenuates pressure overload induced hypertrophy.

Cardiovasc Res 2019 Dec;115(14):1998-2007

Cardiovascular Research Institute, National University Health Systems, MD6 Centre for Translational Medicine, 14 Medical Drive, Singapore, Singapore.

Aims: We and others have previously described the expression landscape of circular RNA (circRNA) in mouse and human hearts. However, the functional relevance of many of these abundantly expressed cardiomyocyte circRNA remains to be fully explored. Among the most abundant circRNA, one stems from the sodium-calcium exchanger gene, Slc8a1, exon 2 locus. Because of its very high abundance in cardiomyocytes we investigated the possible role of circSlc8a1 in the heart.

Methods And Results: We performed a miRNA screen using an array of 752 miRNAs with RNA recovered from a pull-down of endogenous cardiomyocyte circSlc8a1. MicroRNA-133a (miR-133a), with a prior well-recognized role in cardiac hypertrophy, was highly enriched in the fraction of circSlc8a1 pull-down (adjusted P-value < 0.001). We, therefore, followed-up validation of the functional interaction between circSlc8a1 and miR-133 using luciferase assays and reciprocal pull-down assays. In vivo, AAV9-mediated RNAi knockdown of circSlc8a1 attenuates cardiac hypertrophy from pressure-overload, whereas forced cardiomyocyte specific overexpression of circSlc8a1 resulted in heart failure. Molecular analyses showed targets of miR-133a including serum response factor (Srf), connective tissue growth factor (Ctgf), adrenoceptor beta 1 (Adrb1), and adenylate cyclase 6 (Adcy6) to be regulated by circSlc8a1-directed intervention of knockdown and overexpression.

Conclusion: In summary, circSlc8a1 can function as an endogenous sponge for miR-133a in cardiomyocytes. We propose that circSlc8a1 may serve as a novel therapeutic target for cardiac hypertrophy.
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http://dx.doi.org/10.1093/cvr/cvz130DOI Listing
December 2019

Robust CTCF-Based Chromatin Architecture Underpins Epigenetic Changes in the Heart Failure Stress-Gene Response.

Circulation 2019 04;139(16):1937-1956

Genome Institute of Singapore (D.P.L., W.L.W.T., C.G.A.-N., C.J.M.L., C.X.C., Z.T., S.L.N., M.E., M.I.A., S.P., R.S.-Y.F.).

Background: The human genome folds in 3 dimensions to form thousands of chromatin loops inside the nucleus, encasing genes and cis-regulatory elements for accurate gene expression control. Physical tethers of loops are anchored by the DNA-binding protein CTCF and the cohesin ring complex. Because heart failure is characterized by hallmark gene expression changes, it was recently reported that substantial CTCF-related chromatin reorganization underpins the myocardial stress-gene response, paralleled by chromatin domain boundary changes observed in CTCF knockout.

Methods: We undertook an independent and orthogonal analysis of chromatin organization with mouse pressure-overload model of myocardial stress (transverse aortic constriction) and cardiomyocyte-specific knockout of Ctcf. We also downloaded published data sets of similar cardiac mouse models and subjected them to independent reanalysis.

Results: We found that the cardiomyocyte chromatin architecture remains broadly stable in transverse aortic constriction hearts, whereas Ctcf knockout resulted in ≈99% abolition of global chromatin loops. Disease gene expression changes correlated instead with differential histone H3K27-acetylation enrichment at their respective proximal and distal interacting genomic enhancers confined within these static chromatin structures. Moreover, coregulated genes were mapped out as interconnected gene sets on the basis of their multigene 3D interactions.

Conclusions: This work reveals a more stable genome-wide chromatin framework than previously described. Myocardial stress-gene transcription responds instead through H3K27-acetylation enhancer enrichment dynamics and gene networks of coregulation. Robust and intact CTCF looping is required for the induction of a rapid and accurate stress response.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.036726DOI Listing
April 2019

Following hearts, one cell at a time: recent applications of single-cell RNA sequencing to the understanding of heart disease.

Nat Commun 2018 10 30;9(1):4434. Epub 2018 Oct 30.

Cardiovascular Research Institute, National University Health System, 14 Medical Drive, Singapore, 117599, Singapore.

The mammalian heart contains heterogeneous cell types contributing to pathological changes in cardiac disease. In this Comment, we explore how single-cell transcriptomic approaches are unveiling intricate cellular mechanisms and gene co-expression networks that regulate the workings, and failings, of the heart.
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http://dx.doi.org/10.1038/s41467-018-06894-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207674PMC
October 2018

Metformin Inhibits Cellular Proliferation and Bioenergetics in Colorectal Cancer Patient-Derived Xenografts.

Mol Cancer Ther 2017 09 22;16(9):2035-2044. Epub 2017 May 22.

Institute of Bioengineering and Nanotechnology Singapore, Singapore.

There is increasing preclinical evidence suggesting that metformin, an antidiabetic drug, has anticancer properties against various malignancies, including colorectal cancer. However, the majority of evidence, which was derived from cancer cell lines and xenografts, was likely to overestimate the benefit of metformin because these models are inadequate and require supraphysiologic levels of metformin. Here, we generated patient-derived xenograft (PDX) lines from 2 colorectal cancer patients to assess the properties of metformin and 5-fluorouracil (5-FU), the first-line drug treatment for colorectal cancer. Metformin (150 mg/kg) as a single agent inhibits the growth of both PDX tumors by at least 50% ( < 0.05) when administered orally for 24 days. In one of the PDX models, metformin given concurrently with 5-FU (25 mg/kg) leads to an 85% ( = 0.054) growth inhibition. culture of organoids generated from PDX demonstrates that metformin inhibits growth by executing metabolic changes to decrease oxygen consumption and activating AMPK-mediated pathways. In addition, we also performed genetic characterizations of serial PDX samples with corresponding parental tissues from patients using next-generation sequencing (NGS). Our pilot NGS study demonstrates that PDX represents a useful platform for analysis in cancer research because it demonstrates high fidelity with parental tumor. Furthermore, NGS analysis of PDX may be useful to determine genetic identifiers of drug response. This is the first preclinical study using PDX and PDX-derived organoids to investigate the efficacy of metformin in colorectal cancer. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0793DOI Listing
September 2017

The spatial organization of intra-tumour heterogeneity and evolutionary trajectories of metastases in hepatocellular carcinoma.

Nat Commun 2017 02 27;8:4565. Epub 2017 Feb 27.

Division of Surgical Oncology, National Cancer Centre, Singapore 169610, Singapore.

Hepatocellular carcinoma (HCC) has one of the poorest survival rates among cancers. Using multi-regional sampling of nine resected HCC with different aetiologies, here we construct phylogenetic relationships of these sectors, showing diverse levels of genetic sharing, spanning early to late diversification. Unlike the variegated pattern found in colorectal cancers, a large proportion of HCC display a clear isolation-by-distance pattern where spatially closer sectors are genetically more similar. Two resected intra-hepatic metastases showed genetic divergence occurring before and after primary tumour diversification, respectively. Metastatic tumours had much higher variability than their primary tumours, suggesting that intra-hepatic metastasis is accompanied by rapid diversification at the distant location. The presence of co-existing mutations offers the possibility of drug repositioning for HCC treatment. Taken together, these insights into intra-tumour heterogeneity allow for a comprehensive understanding of the evolutionary trajectories of HCC and suggest novel avenues for personalized therapy.
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http://dx.doi.org/10.1038/ncomms14565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333358PMC
February 2017
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