Project description:NAD synthetase 1 (encoded by the gene NADSYN1) is a cytosolic enzyme that catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan and nicotinic acid. NADSYN1 deficiency has recently been added to the spectrum of congenital NAD+ deficiency disorders. To gain insight into the metabolic consequences of NADSYN1 deficiency, the encoding gene was disrupted in A549 and HEK293T cells, and the metabolome was profiled in the presence of different NAD+ precursors, including tryptophan, nicotinamide and nicotinic acid. We demonstrate that when precursors of the NAD+ salvage pathway in the form of nicotinamide become limiting, NADSYN1 deficiency results in a decline in intracellular NAD+ levels even in the presence of other potential NAD+ sources such as tryptophan and nicotinic acid. As a consequence, alterations in 122 and 69 metabolites are observed in NADSYN1-deficient A549 and HEK293T cells compared to the wild-type cell line (FC > 2 and p < 0.05). We thus show that NADSYN1 deficiency results in a metabolic phenotype characterized by alterations in glycolysis, the TCA cycle, the pentose phosphate pathway, and the polyol pathway.
Project description:OPA1, a dynamin-related GTPase mutated in autosomal dominant optic atrophy, is essential for the fusion of the inner mitochondrial membrane. Although OPA1 deficiency leads to impaired mitochondrial morphology, the role of OPA1 in central carbon metabolism remains unclear. Here, we aim to explore the functional role and metabolic mechanism of OPA1 in cell fitness beyond the control of mitochondrial fusion. We applied [U-13C]glucose and [U-13C]glutamine isotope tracing techniques to OPA1-knockout (OPA1-KO) mouse embryonic fibroblasts (MEFs) compared to OPA1 wild-type (OPA1-WT) controls. Furthermore, the resulting tracing data were integrated by metabolic flux analysis to understand the underlying metabolic mechanism through which OPA1 deficiency reprograms cellular metabolism. OPA1-deficient MEFs were depleted of intracellular citrate, which was consistent with the decreased oxygen consumption rate in these cells with mitochondrial fission that is not balanced by mitochondrial fusion. Whereas oxidative glucose metabolism was impaired, OPA1-deficient cells activated glutamine-dependent reductive carboxylation and subsequently relied on this reductive metabolism to produce cytosolic citrate as a predominant acetyl-CoA source for de novo fatty acid synthesis. Prevention of cytosolic glutamine reductive carboxylation by GSK321, an inhibitor of isocitrate dehydrogenase 1 (IDH1), largely repressed lipid synthesis and blocked cell proliferation in OPA1-deficient MEFs. Our data support that, when glucose oxidation failed to support lipogenesis and proliferation in cells with unbalanced mitochondrial fission, OPA1 deficiency stimulated metabolic anaplerosis into glutamine-dependent reductive carboxylation in an IDH1-mediated manner.
Project description:Lynch syndrome is the most common cause of hereditary colorectal cancer and is secondary to germline alterations in one of four DNA mismatch repair (MMR) genes. Here we aimed to provide novel insights into the initiation of MMR-deficient (MMRd) colorectal carcinogenesis by characterizing the expression profile of MMRd intestinal stem cells (ISC). A tissue-specific MMRd mouse model (Villin-Cre;Msh2 LoxP/LoxP ) was crossed with a reporter mouse (Lgr5-EGFP-IRES-creERT2) to trace and isolate ISCs (Lgr5+) using flow cytometry. Three different ISC genotypes (Msh2-KO, Msh2-HET, and Msh2-WT) were isolated and processed for mRNA-seq and mass spectrometry, followed by bioinformatic analyses to identify expression signatures of complete MMRd and haplo-insufficiency. These findings were validated using qRT-PCR, IHC, and whole transcriptomic sequencing in mouse tissues, organoids, and a cohort of human samples, including normal colorectal mucosa, premalignant lesions, and early-stage colorectal cancers from patients with Lynch syndrome and patients with familial adenomatous polyposis (FAP) as controls. Msh2-KO ISCs clustered together with differentiated intestinal epithelial cells from all genotypes. Gene-set enrichment analysis indicated inhibition of replication, cell-cycle progression, and the Wnt pathway and activation of epithelial signaling and immune reaction. An expression signature derived from MMRd ISCs successfully distinguished MMRd neoplastic lesions of patients with Lynch syndrome from FAP controls. SPP1 was specifically upregulated in MMRd ISCs and colocalized with LGR5 in Lynch syndrome colorectal premalignant lesions and tumors. These results show that expression signatures of MMRd ISC recapitulate the initial steps of Lynch syndrome carcinogenesis and have the potential to unveil novel biomarkers of early cancer initiation. SIGNIFICANCE: The transcriptomic and proteomic profile of MMR-deficient intestinal stem cells displays a unique set of genes with potential roles as biomarkers of cancer initiation and early progression.
Project description:Fumarate hydratase (FH) is a mitochondrial enzyme that catalyzes the reversible hydration of fumarate to malate in the tricarboxylic acid (TCA) cycle. Germline mutations of FH lead to hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a cancer syndrome characterized by a highly aggressive form of renal cancer. Although HLRCC tumors metastasize rapidly, FH-deficient mice develop premalignant cysts in the kidneys, rather than carcinomas. How Fh1-deficient cells overcome these tumor-suppressive events during transformation is unknown. Here, we perform a genome-wide CRISPR-Cas9 screen to identify genes that, when ablated, enhance the proliferation of Fh1-deficient cells. We found that the depletion of the histone cell cycle regulator (HIRA) enhances proliferation and invasion of Fh1-deficient cells in vitro and in vivo. Mechanistically, Hira loss activates MYC and its target genes, increasing nucleotide metabolism specifically in Fh1-deficient cells, independent of its histone chaperone activity. These results are instrumental for understanding mechanisms of tumorigenesis in HLRCC and the development of targeted treatments for patients.
Project description:The purpose of this study was to evaluate the effect of progressive weight loss (5, 10, 15% weight loss) on metabolic function such as multi-organ insulin sensitivity and beta-cell function in obese people. We conducted microarray analysis to determine the effect of progressive weight loss on adipose tissue gene expression profile. We examined subcuntaneous adipose tissue samples obtained from 9 obese subjects before (A) and after 5% (B), 10% (C) and 15% (D) weight loss (total 36 samples).
Project description:There is growing evidence about the role of mesenchymal stem cells (MSCs) as cancer stem cells in many sarcomas. Nevertheless, little is still known about the cellular and molecular mechanisms underlying MSCs transformation. We aimed at investigating the role of p53 and p21, two important regulators of the cell cycle progression and apoptosis normally involved in protection against tumorigenesis. Mesenchymal stem cells from wild-type, p21(-/-)p53(+/+), and p21(-/-)p53(+/-) mice were cultured in vitro and analyzed for the appearance of tumoral transformation properties after low, medium, and high number of passages both in vitro and in vivo. Wild-type or p21(-/-)p53(+/+) MSCs did not show any sign of tumoral transformation. Indeed, after short-term in vitro culture, wild-type MSCs became senescent, and p21(-/-)p53(+/+) MSCs showed an elevated spontaneous apoptosis rate. Conversely, MSCs carrying a mutation in one allele of the p53 gene (p21(-/-)p53(+/-) MSCs) completely lost p53 expression after in vitro long-term culture. Loss of p53 was accompanied by a significant increase in the growth rate, gain of karyotypic instability, loss of p16 expression, and lack of senescence response. Finally, these cells were able to form fibrosarcomas partially differentiated into different mesenchymal lineages when injected in immunodeficient mice both after subcutaneous and intrafemoral injection. These findings show that MSCs are very sensitive to mutations in genes involved in cell cycle control and that these deficiencies can be at the origin of some mesodermic tumors.
Project description:Recent evidence indicates that the accumulation of endogenous DNA damage can induce senescence and limit the function of adult stem cells. It remains elusive whether deficiency in DNA damage repair is associated with the functional alteration of mammary stem cells. In this article, we reported that senescence was induced in mammary epithelial cells during aging along with increased expression of p16Ink4a (p16), an inhibitor of CDK4 and CKD6. Loss of p16 abrogated the age-induced senescence in mammary epithelial cells and significantly increased mammary stem cell function. We showed that loss of Brca1, a tumor suppressor that functions in DNA damage repair, in the mammary epithelium induced senescence with induction of p16 and a decline of stem cell function, which was rescued by p16 loss. These data not only answer the question as to whether deficiency in DNA damage repair is associated with the functional decline of mammary stem cells, but also identify the role of p16 in suppressing Brca1-deficient mammary stem cell function.
Project description:Pancreatic ductal adenocarcinoma (PDAC) is a major research focus because of its poor therapy response and dismal prognosis. PDAC cells adapt their metabolism to the surrounding environment, often relying on diverse nutrient sources. Because traditional experimental techniques appear exhaustive to find a viable therapeutic strategy, a highly curated and omics-informed PDAC genome-scale metabolic model was reconstructed using patient-specific transcriptomics data. From the model-predictions, several new metabolic functions were explored as potential therapeutic targets in addition to the known metabolic hallmarks of PDAC. Significant downregulation in the peroxisomal beta oxidation pathway, flux modulation in the carnitine shuttle system, and upregulation in the reactive oxygen species detoxification pathway reactions were observed. These unique metabolic traits of PDAC were correlated with potential drug combinations targeting genes with poor prognosis in PDAC. Overall, this study provides a better understanding of the metabolic vulnerabilities in PDAC and will lead to novel effective therapeutic strategies.