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MTCH2) Suppresses Tumor

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MTCH2) Suppresses Tumor 1 Inhibition of Mitochondrial Carrier Homolog 2 (MTCH2) Suppresses Tumor 2 Invasion and Enhances Sensitivity to Temozolomide in Malignant Glioma 3 4 Authors: Qiuyun Yuan1,#, Wanchun Yang1,#, Shuxin Zhang1, Tengfei Li1, Mingrong Zuo1, 5 Xingwang Zhou1, Junhong Li1, Mao Li1, Xiaoqiang Xia1, Mina Chen1,*, Yanhui Liu1,* 6 7 Affiliation: 8 1Department of Neurosurgery, State Key Laboratory of Biotherapy, West China Hospital, Sichuan 9 University, Chengdu 610041, People’s Republic of China. 10 #These authors contributed equally to this work. 11 12 *Correspondence: Mina Chen ([email protected]) and Yanhui Liu ([email protected]), 13 Department of Neurosurgery, West China Hospital, Sichuan University, 37 Guoxue Avenue, 14 Chengdu, 610041, Sichuan Province, People’s Republic of China. - 1 - 15 Abstract 16 Background: Malignant glioma exerts a metabolic shift from oxidative phosphorylation 17 (OXPHOs) to aerobic glycolysis, with suppressed mitochondrial functions. This phenomenon 18 offers a proliferation advantage to tumor cells and decrease mitochondria-dependent cell death. 19 However, the underlying mechanism for mitochondrial dysfunction in glioma is not well 20 elucidated. MTCH2 is a mitochondrial outer membrane protein that regulates mitochondrial 21 metabolism and related cell death. This study aims to clarify the role of MTCH2 in glioma. 22 Methods: Bioinformatic analysis from TCGA and CGGA databases were used to investigate the 23 association of MTCH2 with glioma malignancy and clinical significance. The expression of 24 MTCH2 was verified from clinical specimens using real-time PCR and western blots in our 25 cohorts. siRNA-mediated MTCH2 knockdown were used to assess the biological functions of 26 MTCH2 in glioma progression, including cell invasion and temozolomide-induced cell death. 27 Biochemical investigations of mitochondrial and cellular signaling alternations were performed 28 to detect the mechanism by which MTCH2 regulates glioma malignancy. 29 Results: Bioinformatic data from public database and our cohort showed that MTCH2 30 expression was closely associated with glioma malignancy and poor patient survival. Silencing 31 of MTCH2 expression impaired cell migration and enhanced temozolomide sensitivity of human 32 glioma cells. Mechanistically, MTCH2 knockdown increased mitochondrial oxidative damage 33 and decreased pro-survival AKT signaling. 34 Conclusion: Our work identifies the oncogenic role of MTCH2 in gliomas, and establishes the 35 causal relationship between MTCH2 expression and glioma malignancy, which may provide a 36 potential target for future interventions. 37 Key words: MTCH2, glioma, temozolomide, mitochondria, cell migration, cell death. - 2 - 38 Abbreviations 39 MTCH2: mitochondrial carrier homolog 2; 40 GBM: glioblastoma; 41 TMZ: temozolomide; 42 OXPHOs: oxidative phosphorylations; 43 TCGA: The Cancer Genome Atlas Research Network; 44 CGGA: the Chinese Glioma Genome Atlas; 45 4-HNE: 4-Hydroxy-2-Nonenal; 46 ROS: reactive oxygen species. - 3 - 47 Background 48 Originating from glial cells, glioma is a common type of malignant and lethal tumor in the brain, 49 which can be divided into astrocytomas, oligodendrogliomas, and glioblastomas (GBM) (1, 2). 50 Glioma displays aggressive growth and diffuse invasion, and evolves rapidly by acquiring new 51 mutations and oncogene expressions for drug resistance (3). Although current standard care of 52 maximal safe surgical resection followed by radiotherapy and concurrent temozolomide (TMZ) 53 chemotherapy provides survival benefit to selected patients (4), glioma still exhibits poor 54 responses to approved therapies and carries an inferior prognosis. Therefore, molecular 55 understandings of the initiation and progression of glioma are urgently needed. 56 Over the last decades, a well-appreciated hallmark of glioma is the altered tumor metabolism 57 (5, 6), a phenomenon known as Warburg effect (7). This effect fulfills the demands for energy 58 and biomass production of rapid proliferation in glioma cells (6). Extensive studies established 59 that mitochondrial dysfunction is a prominent mechanism by which glioma cells exert its 60 metabolic shift from oxidative phosphorylation (OXPHOs) to glycolysis, regardless of oxygen 61 availability. During this metabolic reprogramming, mitochondrial function is glioma is evidently 62 suppressed through mutations of mitochondrial DNA (8), altered metabolic enzyme atlas (9, 10), 63 and imbalanced morphological dynamics (11). All these events offer a proliferation advantage to 64 tumor cells and decrease mitochondria-dependent cell death to chemical drugs (12). Although a 65 number of studies have increased the understanding of mitochondrial dysfunction in glioma cells, 66 the molecular repertoire is still largely unknown. 67 To explore the mechanistic interplay between mitochondrial dysfunction and glioma 68 progression, we focused on mitochondrial carrier homolog 2 (MTCH2), a critical regulator of 69 mitochondrial metabolism and related cell death (13, 14). MTCH2 is a mitochondrial outer - 4 - 70 membrane protein that interacts with the truncated BH3-interacting domain death agonist (tBID) 71 to regulate cell apoptosis (15-17). Previous studies demonstrate that loss of MTCH2 impairs 72 mitochondrial architecture and functions, including enlarged size (18), reduced motility (19), and 73 elevated oxidative stress (20). MTCH2 expression was associated with several types of tumors. 74 For example, MTCH2 was down-regulated in ErbB2-driven mammary carcinoma (21), and its 75 induction reduced tumorigenicity and leads to growth arrest of breast cancer (22). However, a 76 recent study showed the oncogenic function of MTCH2 in acute myeloid leukemia (AML). 77 Inhibiting MTCH2 decreased tumor growth and induced differentiation of AML cells (23). 78 However, the role of MTCH2 in malignant glioma is not defined. 79 In this study, we reveal the oncogenic function of MTCH2 in malignant glioma. MTCH2 80 expression is closely associated with glioma progression, and its knockdown impairs cell 81 migration and enhances temozolomide sensitivity of glioma cells. Our work establishes the 82 causal relationship between MTCH2 expression and glioma malignancy, and defines MTCH2 as 83 a potential target for future interventions. 84 85 Methods 86 Patient samples 87 Glioma and adjacent brain tissues included in this study were obtained from primary glioma 88 patients undergoing elective craniotomy at West China Hospital between January 2019 and 89 January 2020. All human studies were approved by the Institutional Review Board of West 90 China Hospital of Sichuan University (project identification code: 2018.569). All patients 91 provided written informed consent. The diagnoses of glioma grade were made by three 92 independent pathologists. Clinical samples were used for detection the mRNA and protein levels. - 5 - 93 Bioinformatic analysis 94 Biomarker data and clinical information of The Cancer Genome Atlas Research Network (TCGA) 95 and the Chinese Glioma Genome Atlas (CGGA) LGG-GBM cohorts were downloaded from 96 TCGA (https://gdc-portal.nci.nih.gov/) and CGGA (http://www.cgga.org.cn) repository. IDH 97 mutation was determined by merging IDH1 codon 132 and IDH2 codon 172 mutations. 98 Chromosome 1p19q co-deletion was determined by 1p32 and 19q13 segment focal CNV value 99 less than -0.3. MGMT promoter methylation level bins were divided using the 10% and 40% 100 percentiles of mean beta value of probes between 131264896 and 131265737 on chromosome 10 101 (GRCh38 genome). For survival analysis, patients were divided into low and high gene 102 expression groups by median expression level. Hazard ratio for expression was tested using log 103 rank test. Gene expression is presented as normalized FPKM. Correlation and survival analysis 104 were conducted in R using grid and survminer packages. 105 106 Cell culture and siRNAs transfection 107 Human glioblastoma cell lines U-87MG, U-251 MG and A172 cell lines were obtained from 108 Type Culture Collection of the Chinese Academy of Sciences (CTCC, Shanghai, China). Glioma 109 cells were maintained at DMEM (Hyclone) with 10% FBS (PAN), and cultured under 5% CO2 at 110 37°C incubator. All cells were validated by Short Tandem Repeat (STR) analysis. For MTCH2 111 knockdown, targeted siRNA were transfected into cultured glioma cells using Lipofectamine 112 RNAiMAX reagent (Invitrogen) in Opti-MEM (Invitrogen) over 72 hours. The siRNA sequences 113 are as follow: siRNA MTCH2#1: sense 5'-GGUAAAGUUUUACAGCAUUTT-3'; antisense 114 5'-AAUGCUGUAAAACUUUACCAT-3'; siRNA MTCH2#2: sense 115 5'-GAGCCGAGGAAAUAGCUUATT-3'; antisense 5'- UAAGCUAUUUCCUCGGCUCAT-3'. - 6 - 116 Immunofluorescence 117 For immunofluorescence assay to observe MTCH2 cellular localization, A172 cells were 118 cultured on coverslips over 48 hours. For Mito-Tracker staining, A172 cells were incubated with 119 the Mito-Tracker dye (Invitrogen, 200 nM) in DMEM at 37°C incubator for 15 minutes (mins). 120 Then, cells were fixed with 4% paraformaldehyde (PFA, Sigma) solution at room temperature for 121 30 mins. After permeabilization with 0.25% Triton X-100 in PBS, cells were blocked with 10% 122 goat serum and incubated with primary antibodies (anti-MTCH2, Proteintech) overnight at 4°C. 123 Then cells were washed with PBS for three times and incubated with ALEXA FLUOR 488/594 124 secondary antibodies, and mounted slides using ProLong® Gold Antifade Reagent with DAPI 125 (Invitrogen). Images were acquired using Olympus BX63 microscope. 126
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