Integrated metabolomics and lipidomics analyses reveal metabolic

Subscriber access provided by Iowa State University | Library

Article

Integrated metabolomics and lipidomics analyses reveal metabolic reprogramming in human glioma with IDH1 mutation Lina Zhou, Zhichao Wang, Chunxiu Hu, Chaoqi Zhang, Petia Kovatcheva-Datchary, Di Yu, Shasha Liu, Feifei Ren, Xiaolin Wang, Yanli Li, Xiaoli Hou, Hailong Piao, Xin Lu, Yi Zhang, and Guowang Xu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00663 • Publication Date (Web): 31 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32

Journal of Proteome Research

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Paragon Plus Environment

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 32

Integrated metabolomics and lipidomics analyses reveal metabolic reprogramming in human glioma with IDH1 mutation

Lina Zhou1, ▲, Zhichao Wang1,3, ▲, Chunxiu Hu1, Chaoqi Zhang2, Petia Kovatcheva-Datchary 1, Di Yu1,3, Shasha Liu2, Feifei Ren2, Xiaolin Wang1,Yanli Li1, Xiaoli Hou1, Hailong Piao1, Xin Lu1, Yi Zhang2,*, Guowang Xu1,*

1 CAS

Key Laboratory of Separation Science for Analytical Chemistry, Dalian

Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. 2

Biotherapy Center and Cancer Center,The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China

3

University of Chinese Academy of Sciences, Beijing 100049, China.

▲: contributed

equally to this paper.

Corresponding Authors: *Guowang Xu, CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Telephone: +86-411-84379530; Fax: +86-411-84379559; E-mail: [email protected]； Yi Zhang, Biotherapy Center and Cancer Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China. E-mail: [email protected].

1


Page 3 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT Mutations in isocitrate dehydrogenase (IDH) 1 are high-frequency events in low grade glioma and secondary glioblastoma and IDH1 mutant glioma are vulnerable to interventions. Metabolic reprogramming is a hallmark of cancer. In this study, comprehensive metabolism investigation of clinical IDH1 mutant glioma specimens was performed to explore its specific metabolic reprogramming in real microenviroment. Massive metabolic alterations from glycolysis to lipid metabolism were identified in the IDH1 mutant glioma tissue when compared to IDH1 wild-type glioma. Of note, TCA cycle intermediates were in similar levels in both groups, having more pyruvate found entering TCA cycle in IDH1 mutant glioma. The pool of fatty acyl chains was also reduced, displaying as decreased triglycerides and sphingolipids, though membrane phophatidy lipids were not changed. The lower fatty acyl pool may be mediated by the lower protein expression levels of long-chain acyl-CoA synthetase 1 (ACSL1), ACSL4 and very long-chain acyl-CoA synthetase 3 (ACSVL3) in IDH1 mutant glioma. Lower ACSL1 was further found contributing to the better survival of IDH1 mutant glioma patients based on the The Cancer Genome Atlas (TCGA) RNA sequencing data. Our research provides valuable insights in the tissue metabolism of human IDH1 mutant glioma and unravels new lipid-related targets.

KEY WORDS: glioma, IDH1, metabolomics, lipidomics, metabolic reprogramming

2



Page 4 of 32

INTRODUCTION Glioma is the most common malignant brain tumors with significant mortality and morbidity 1. It has been reported that isocitrate dehydrogenase (IDH) 1 mutation on amino acid 132 occurs in more than 70% of the low grade gliomas and secondary glioblastomas on the basis of histopathological and clinical criteria established by the World Health Organization (WHO)

2-4.

Wild-type IDH catalyzes the oxidative

decarboxylation of isocitrate to alpha-ketoglutarate (2-KG), producing nicotinamide adenine dinucleotide phosphate (NADPH) 5, a cofactor whose function is to maintain normal levels of reduced glutathione and rescue the cell from reactive oxygen species (ROS) 6. Mutant IDH loses the wild-type enzyme function, but acquires a new ability to catalyze the reduction of 2-KG to 2-hydroglutarate (2-HG), consuming NADPH 7-9. Thus, with the significant production of 2-HG, IDH mutation may present an increased oxidative stress and great metabolic stress in maintaining tricarboxylic acid cycle (TCA) and molecular synthesis for cell proliferation 10. In the recent years, IDH mutation has been found closely related to oncogenesis either through induction of hypoxia inducible factor-1 pathway

11,

or contributing to tumorigenesis and tumor

progression through producing the oncometabolite 2-HG, resulting in histone and DNA hypermethylation

7-9.

However, patients with IDH1 or IDH2 mutations were

reported to have a longer survival than those without IDH mutation 4. The exact roles of IDH mutations in the occurrence and the progression of glioma are still not clear. Metabolic reprogramming is one of the hallmarks of cancer mutation,

somatic

mutations

in

other

two

metabolic

12.

Except for IDH

enzymes,

succinate

dehydrogenase (SDH) and fumarate hydratase (FH), have been found associated with tumorigenesis 13. Also, metabolic reprogramming is regulated by oncogenic signaling 3


Page 5 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


to aid cell growth and proliferation

14-15.

Thus, except for 2-HG production, other

metabolic changes may also be related to tumor progression. To investigate the metabolic characteristics of IDH1 mutant glioma, some researches have been performed by investigating isogenic IDH1-R132H mutant cell models, cultured under normoxia 16-20. Most of the differential amino acids were accumulated, but glutamate, aspartate and intermediates in the late TCA cycle were decreased in IDH1-R132 mutant cells and 2-HG treated cells, together with increased glycerol phosphocholine, but decreased phosphocholine 16. Decreased glutamate and phosphocholine were also reported in IDH1 mutant cells, together with lactate

17.

Lactate dehydrogenase

(LDHA) activity was found lower in IDH1 mutant glioma cells, and silencing of the LDHA was associated with increased methylation of the LDHA promoter 20. Glucose flux to TCA cycle was reduced through pyruvate dehydrogenase (PDH) activity in U87 glioblastoma and NHA-IDH1 mutant cells, which was associated with increased PDH inhibitory phosphorylation regulated by pyruvate dehydrogenase kinase-3

18-19.

Further, CE-MS based metabolomics analysis of human glioma tissue revealed that N-acetylated amino acids, glutamine and glutamate were decreased in 13 IDH1 wild-type glioma tissues and 20 IDH1-R132H mutant glioma tissues 21. Another study on animal and human gliomas also reported lower level of phosphocholine and higher glycerol phosphocholine in IDH1-R132H mutant glioma monitored by resolution magic angle spinning spectroscopy

22.

31P

high

Recently, using mass spectrometry

imaging on patient-derived xenografts, the metabolic landscape in IDH-mutant glioma versus wild type glioma has been shown to have phospholipid, energy, and oxidative stress pathways affected

23.

Collectively, increasing metabolic features have been

provided, which will increase the understanding of IDH1 mutant glioma malignant progression. But still more systematic investigations of the metabolic consequences of 4



Page 6 of 32

IDH1 mutation need to be performed in human glioma tissues, which reflect the metabolic reprogramming in response to the real tissue microenvironment. Metabolomics enables a comprehensive monitoring of the responses of endogenous metabolites towards pathophysiological stimuli including genetic alterations and disturbances in metabolic activities

24.

Here we performed gas

chromatography (GC)-mass spectrometry (MS) and liquid chromatography (LC)-MS based metabolomics and LC-MS based lipidomics analyses to identify important metabolic alterations in glioma tissues with and without IDH1 mutations. Key related proteins were also analyzed to explore important targets responsible for the key metabolic alterations in human glioma tissues with IDH1 mutation.

EXPERIMENTAL SECTION Subjects Seventy-five glioma patients in the first affiliated hospital of Zhengzhou University were recruited for the retrospective study. Brain tumor tissue from all glioma patients and adjacent tissue from 41 of the patients were collected during surgery. Samples of grossly normal adjacent brain tissues were removed as far from the tumor as the operative field permitted. Tissue samples were immediately kept on ice within one hour and then stored at -80 ˚C until analysis. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Scientific Research and Clinical Trial Ethics Committee of the First Affiliated Hospital of Zhengzhou University.

5


Page 7 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Glioma histopathological and molecular classification All glioma tissues were subjected to consensus review by at least two neuropathologists and graded according to histopathological and clinical criteria established by the WHO. Genomic DNA was isolated from the brain tumor samples using the Animal Tissues/Cells Genomic DNA Extraction kit (Solarbio, Beijing, China), following the manufacture protocol. The assessments of IDH1 and IDH2 mutations in the glioma tissues were performed by Tsingke Biotech Co., Ltd. (Beijing, China) with a 2720 Thermal cycler (Applied Biosystems, USA) using the following primer sets: IDH1-F: 5`-AAT GAG CTC TAT ATG CCA TCA CTG-3`; IDH1-R:5`-TTC ATA CCT TGC TTA ATG GGT GT-3`; IDH2-F: 5`- CCC GTC TGG CTG TGT TGT T-3`; IDH2-R: 5`-TCC TTG ACA CCA CTG CCAT-3`.

Metabolomics and lipidomics analysis The tissue samples were not thawed until sample preparation for metabolomics and lipidomics analysis. The procedure for GC-MS and LC-MS based metabolomics, LC-MS based lipidomics analysis, including sample preparation, data acquisition and the following data preprocessing are provided in the supplemental materials.

Statistical analyses The multivariate partial least-squares discriminant analysis (PLS-DA) was performed employing SIMCA-P 11.0 (Umetrics, Sweden) with all variables unit variance scaled. And permutation test was exerted 200 times to see whether the constructed model was overfitted. Paired sample t-test was performed for comparison of paired cancer tissue and adjacent tissue. Pathway analysis was performed on metaboAnalyst 4.0 (http://www.metaboanalyst.ca), the important pathways were defined as having 6



Page 8 of 32

-log(p)>3 and pathway impact factor>0.1. To explore the important metabolic differences of glioma tissues with and without IDH1 mutations, Mann-Whitney U tests, significance analysis of microarrays (SAM) and heat map analysis of significant metabolites

were

performed

using

the

Multi

Experiment

Viewer

(http://www.tm4.org). The level significance for the univariate analysis was set at p

Integrated metabolomics and lipidomics analyses reveal metabolic

Recommend Documents