Citations

Abstract FoxM1, a member of the Forkhead transfection factors, has been found to be overexpressed in many human cancers including ovarian cancer. Although FoxM1 is known to be downregulated by p53, the molecular mechanisms are not well characterized. In this study, we used the MDM2 inhibitor Nutlin-3 as a tool to modulate p53 expression and investigated the mechanism by which p53 regulates FoxM1 expression in ovarian cancer cells. We observed that Nutlin-3 upregulated p53 protein and downregulated FoxM1 mRNA and protein levels in ovarian cancer cell lines with wild type TP53, but not in cell lines with mutant TP53. This effect was at least partially blocked by cycloheximide or actinomycin D, indicating a transcription- and translation-dependent mechanism. Pulse-chase studies revealed that downregulation of FoxM1 by Nutlin-3 is due to enhanced FoxM1 mRNA decay. Knockdown of p53 using shRNAs abrogated the suppression of FoxM1 expression by Nutlin-3, indicating a p53-dependent mechanism. Furthermore, silencing of FoxM1 by shRNAs in ovarian cancer cells with or without wild-type TP53 led to a significant decrease in cell proliferation. The FoxM1 inhibitor, thiostrepton, induces cytotoxicity in ovarian cancer cell lines and enhances sensitivity to cisplatin in these cells. Thiostrepton downregulates FoxM1 expression in several ovarian cancer cell lines and enhances sensitivity to carboplatin in vivo. Finally, FoxM1 expression is elevated in nearly all (47/48) ovarian tumors. In summary, the present study provides novel evidence that p53 downregulates FoxM1 in ovarian cancer cells at least in part by destabilizing FoxM1 mRNA and that FoxM1 may be targeted for therapeutic benefits in ovarian cancer. Citation Format: Xuan Zhang, Lihua Cheng, Kay Minn, Magdalena Waggoner, Nikhil K. Parelkar, Iman Jokar, George A. Vielhauer, Rashna Madan, Andrew K. Godwin, Viji Shridhar,, Jeremy Chien. Targeting p53-FoxM1 axis in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1409.

How endurance training alters muscle lipid metabolism while preserving insulin sensitivity remains unclear. Because acute free fatty acid (FFA) elevation by lipid infusion reduces insulin sensitivity, we hypothesized that training status would alter accumulation of muscle triacylglycerol (TAG), diacylglycerol (DAG), ceramide, and acylcarnitine during acute FFA elevation. Trained ( n = 15) and sedentary ( n = 13) participants matched for age, sex, and BMI received either a 6-h infusion of lipid (20% Intralipid at 90 ml/h) or glycerol (2.25 g/100 ml at 90 ml/h) during a hyperinsulinemic euglycemic clamp. Muscle biopsies were taken at 0, 120, and 360 min after infusion initiation to measure intramyocellular concentrations of TAG, DAG, ceramides, and acylcarnitines by liquid chromatography-tandem mass spectrometry. Trained participants had a higher V̇o2 maxand insulin sensitivity than sedentary participants. The lipid infusion produced a comparable elevation of FFA (594 ± 90 μmol/l in trained, 721 ± 30 μmol/l in sedentary, P = 0.4) and a decline in insulin sensitivity (−44.7% trained vs. −47.2% sedentary, P = 0.89). In both groups, lipid infusion increased the linoleic and linolenic acid content of TAG without changing total TAG. In the sedentary group, lipid infusion increased total, oleic, and linoleic acid and linolenic acid content of DAG. Regardless of training status, lipid infusion did not alter total ceramide, saturated ceramide, palmitoyl-carnitine, or oleoyl-carnitine. We conclude that during acute FFA elevation, trained adults have a similar decline in insulin sensitivity with less accumulation of muscle DAG than sedentary adults, suggesting that lipid-induced insulin resistance can occur without elevation of total muscle DAG.

Significance Cancer cells reprogram their metabolism for optimal growth and survival. AMPK-activated protein kinase (AMPK) is a key energy sensor that controls many metabolic pathways including metabolic reprogramming. However, its role in cancer is poorly understood. Some studies claim that it has a tumor suppressor role while others show its protumor role. Two AMPK-activating compounds (including metformin, now in many clinical trials) are widely used to suppress cancer cell proliferation. We found that AMPK is abundantly expressed in high-grade gliomas and, in contrast to popular belief, these two AMPK activators suppressed glioma cell proliferation through unique AMPK-independent mechanisms.

Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids such as ceramides (Cer) and long-chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity, and at mitochondrial levels it stimulates reactive oxygen species production. Here, we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with a concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%Hb A1c), and muscular LCFa-CoA content. The alternations were accompanied by an increase in protein expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), a decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1), and inhibition of insulin signaling cascade by Aktα and GYS3β phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed, except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression, and phosphorylation status of Akt and GYS3β when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearson's correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoAs and Cers containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, or proteins implicated in Cer synthesis and muscle insulin sensitivity. These persistent changes might contribute to muscle insulin resistance observed in T1D individuals.

RATIONALE Sphingolipids are important components of cell membranes that serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. De novo ceramide biosynthesis depends on fatty acid availability, but whether muscle uses circulating free fatty acids or pre‐existing intracellular stores is unknown. Our goal was to develop a method to detect the incorporation of intravenously infused [U‐ 13 C]palmitate into intramyocellular ceramides. METHODS We used liquid chromatography/tandem mass spectrometry (LC/MS/MS) to measure the concentrations of different sphingolipid species and 13 C‐isotopic enrichment of 16:0‐ceramide. Chromatographic separation was performed using ultra‐performance liquid chromatography. The analysis was performed on a triple quadrupole mass spectrometer using a positive ion electrospray ionization source with selected reaction monitoring (SRM). RESULTS The sphingolipids ions, except enriched ceramide, were monitored as [M+2+H] + . The [ 13 C 16 ]16:0‐ceramide was monitored as [M+16+H] + . By monitoring two different transitions of the [ 13 C 16 ]16:0‐ceramide (554/536 and 554/264) we could indirectly measure enrichment of the palmitate that is not a part of the sphingoid base. Concentration and enrichment could be measured using 20 mg of muscle obtained from volunteers receiving a low dose [U‐ 13 C]palmitate infusion. CONCLUSIONS LC/MS/MS can be used to detect the incorporation of plasma palmitate into muscle ceramides in humans, in vivo . Copyright © 2012 John Wiley & Sons, Ltd.

Insulin regulates many cellular processes, but the full impact of insulin deficiency on cellular functions remains to be defined. Applying a mass spectrometry–based nontargeted metabolomics approach, we report here alterations of 330 plasma metabolites representing 33 metabolic pathways during an 8-h insulin deprivation in type 1 diabetic individuals. These pathways included those known to be affected by insulin such as glucose, amino acid and lipid metabolism, Krebs cycle, and immune responses and those hitherto unknown to be altered including prostaglandin, arachidonic acid, leukotrienes, neurotransmitters, nucleotides, and anti-inflammatory responses. A significant concordance of metabolome and skeletal muscle transcriptome–based pathways supports an assumption that plasma metabolites are chemical fingerprints of cellular events. Although insulin treatment normalized plasma glucose and many other metabolites, there were 71 metabolites and 24 pathways that differed between nondiabetes and insulin-treated type 1 diabetes. Confirmation of many known pathways altered by insulin using a single blood test offers confidence in the current approach. Future research needs to be focused on newly discovered pathways affected by insulin deficiency and systemic insulin treatment to determine whether they contribute to the high morbidity and mortality in T1D despite insulin treatment.