Mitochondrial protein ATPase family, AAA domain containing 3A correlates with radioresistancy in glioblastoma

Abstract

背景：ATPase family, AAA domain containing 3A (ATAD3A)是一粒線體蛋白，被發現常在癌症病人表現並與癌症病人的藥物抗性有關。ATAD3A位於染色體1p36.33，可能與染色體1p刪除所引發的輻射以及化學抗性有關。染色體1p刪除常見於腦瘤病人，因此，本研究主要是探討ATAD3A於腦瘤病人的相關性，以及輻射藥物抗性之研究。 研究方法：我們使用免疫組織染色法(IHC)測定ATAD3A在多型性腦膠質瘤檢體與細胞株的的表現量。比較ATAD3A表現量高低與多型性腦膠質瘤病人存活時間這兩個因子是否有統計上顯著相關。此外，藉由靜默(silence)與促表(enforce expression)ATAD3A的基因進一步分析該基因與輻射抗性的分子生物機轉。 結果：本研究在臨床三十二位多型性腦膠質瘤病人的結果顯示，高粒線體蛋白ATAD3A的免疫組織染色表現有較差的預後，並且是一獨立於MGMT基因的預後因子。在基礎研究方面，靜默高ATAD3A基因表現的GBM細胞株T98G會降低其輻射抗性。相反地，促強低ATAD3A基因表現的GBM細胞株UM87MG會提高輻射抗性。進一步結果顯示，靜默ATAD3A基因會阻礙DNA基因修復，其機轉與ATM修復蛋白無法進入細胞核與後續無法啟動H2AX蛋白有關。更進一步的研究顯示，靜默ATAD3A基因會導致持續性PARP1蛋白的啟動，也提升了靜默ATAD3A基因啟動所導致癌細胞的第三類壞死性死亡的可能性。 結論：我們的研究結果顯示，在臨床上高ATAD3A 表現是一個獨立性地、高輻射抗性的多型性腦膠質瘤表現型(phenotype)。ATAD3A在臨床上極具價值，可做為在治療上的標的。Background: Overexpression of the ATPase family, AAA domain containing 3A (ATAD3A) has been frequently detected in cancer patients and seen to be involved in drug resistance. ATAD3A is located on human chromosome 1p36.33 and may associates with radio- and chemo- sensitivity as a result of deletion of chromosome 1p. This study was conducted to investigate the significance of ATAD3A in GBM. Methods: Expression of ATAD3A in GBM cells was determined by immunohistochemistry. The relationship between ATAD3A expression and patient survival was statistically evaluated. In vitro correlation between ATAD3A and radiation resistance was estimated by silencing or enforced expression of the gene. Results: Clinically, ATAD3A overexpression correlated with worse prognosis and was independent from MGMT methylation status in thirty two GBM patients. In vitro, silencing of ATAD3A reduced radiation resistance in T98G cells. On the other hand, enforced ATAD3A expression increased radiation resistance in U87MG cells. Surprisingly, knockdown of ATAD3A (ATAD3Akd) augmented DNA repairs by interfering nuclear translocation of ATM and subsequent phosphorylation of H2AX. Moreover, sustained over-activation of PARP of ATAD3Akd before and after irradiation also raised the possibility of type III necrotic cell death Conclusion: Our data suggested that overexpression of ATAD3A is an independent phenotype of radioresistance in patients with GBM, and ATAD3A could be a potential target for therapy.Abstract (Chinese) I Abstract (English) III Table of contents IV List of figures and tables VI Chapter 1 Introduction of glioblastoma and Literature reviews p.1 (1) Introduction p.1 (2) Epidemiology p.2 (3) Clinical manifestations p.2 (4) Current standard of care p.3 (5) Prognostic factors and survival p.5 (6) GBM subtypes and gene expression-based molecular classification p.6 (7) Genetic alterations of GBMs p.8 (8) Specific molecular biomarkers –MGMT p.10 (9) New targeted therapies p.11 Chapter 2 Material and Methods p.15 1. Materials p.15 (1) Cell line p.15 (2) GBM specimen p.15 (3) Antibodies p.15 (4) Primer and probe p.16 (5) Software p.16 2. Methods p.16 (1) Preparation and characterization of mouse polyclonal and monoclonal antibodies p.16 (2) Immunoblotting analysis p.17 (3) Lentiviral infection p.18 (4) Immunohistochemistry p.19 (5) Indirect immunofluorescence and confocal microscopy p.19 (6) Colony-Forming Assay p.20 (7) Cell cycle analysis by flow cytometry p.21 (8) Electron microscopy p.21 (9) Methylation-specific QPCR p.22 (10) γ-H2AX Assay p.22 (11) Gel Electrophoretic Analysis of DNA Double-Stranded Breaks p.23 (12) Statistical Analysis p.24 Chapter 3 Results p.25 1. Overexpression of ATAD3A in GBM Specimens as Determined by Immunohistochemistry and Immunoblotting p.25 2. Identification and Validation of ATAD3A Expression in GBM cells p.26 3. Silencing of ATAD3A Expression Increases Radiation Sensitivity and Ectopic Expression of ATAD3A Increases Radiation Resistance p.27 4. The Cytologic Effect of ATAD3A Expression on Radiation-Induced Cell Death p.28 5. Silencing of ATAD3A Down-regulates Expression Levels of DNA Repair-Related Genes and Reduces Nuclear H2AX as well as ATM p.30 Chapter 4 Discussion p.31 Chapter 5 Conclusion p.35 Figures p.36 Tables p.64 Reference p.6