학위논문(박사) -- 서울대학교대학원 : 약학대학 협동과정 천연물과학전공, 2023. 2. 박성혁.Despite growing evidence for mitochondrias involvement in cancer, the roles of specific metabolic components outside the respiratory complex have been little explored. We conducted metabolomic studies on mitochondrial DNA (mtDNA)-deficient (ρ0) cancer cells with lower proliferation rates to clarify the undefined roles of mitochondria in cancer growth. Despite extensive metabolic downregulation, ρ0 cells exhibited high glycerol-3-phosphate (G3P) level, due to low activity of mitochondrial glycerol-3-phosphate dehydrogenase (GPD2). Knockout (KO) of GPD2 resulted in cell growth suppression as well as inhibition of tumor progression in vivo. Surprisingly, this was unrelated to the conventional bioenergetic function of GPD2. Instead, multi-omics results suggested major changes in ether lipid metabolism, for which GPD2 provides dihydroxyacetone phosphate (DHAP) in ether lipid biosynthesis. GPD2 KO cells exhibited significantly lower ether lipid level, and their slower growth was rescued by supplementation of a DHAP precursor or ether lipids. Mechanistically, ether lipid metabolism was associated with Akt pathway, and the downregulation of Akt/mTORC1 pathway due to GPD2 KO was rescued by DHAP supplementation. Overall, the GPD2-ether lipid-Akt axis is newly described for the control of cancer growth. DHAP supply, a non-bioenergetic process, may constitute an important role of mitochondria in cancer.현재에는, 미토콘드리아가 암의 발생 및 생장에 기여한다는 여러 연구 결과가 늘어나고 있음에도 불구하고, 그 연구의 대부분은 미토콘드리아의 호흡 기전과 연관된 역할 및 기능이 대부분을 차지하고 있다. 본 연구에서는, 암 생장에 있어서의 아직 정의되지 않은 미토콘드리아의 역할을 밝혀내기 위하여 미토콘드리아 DNA (mtDNA)가 손실된 ρ0 암세포를 모델로 하여 대사 연구를 수행했다. ρ0 세포에서는 대부분의 대사 활성이 낮아졌음에도 불구하고 높은 수준의 glycerol-3-phosphate (G3P)의 축적을 보여주었고, 이는 미토콘드리아 효소인 mitochondrial glycerol-3-phosphate dehydrogenase (GPD2)의 낮은 발현과 활성으로부터 기인하는 것이었다. 4T1 wildtype (WT) 마우스 유방암 세포에서의 GPD2의 knockout (KO)는, in vitro에서의 암세포의 성장 및 해당 세포로부터 유래한 in vivo 마우스 종양의 성장을 감소시켰다. 이러한 GPD2에 의한 암 억제 현상은, 예상과 다르게, 기존에 알려져 있던 GPD2의 생체 에너지 대사 조절 기전과는 관련이 없었다. 대신에, 멀티오믹스 분석 결과는, GPD2가 dihydroxyacetone phosphate (DHAP)를 생성함으로써 에테르 지질 생합성에 크게 기여한다는 것을 밝혀냈다. GPD2 KO 암세포는 DHAP 및 에테르 지질 수준이 현저히 낮았고, DHAP 또는 에테르 지질의 보충은 GPD2 KO 암세포의 성장을 일부 회복시켜주었다. 더 나아가, 에테르 지질 대사는 PI3K/Akt 신호전달 경로와 연관이 있었고, 이를 통해 암세포의 성장을 조절하는 것으로 보여졌다. 결론적으로, 본 연구에서는 암 성장에 있어서 미토콘드리아의 비 에너지 대사적 동화 작용인 GPD2-ether lipid-Akt 기전을 새로이 제시하고, 더 나아가 이는 곧 암의 치료에 있어서 GPD2가 새로운 치료 표적이 될 수 있다는 가능성을 시사한다.1. Introduction 1
1.1 Mitochondria's role in cancer 1
1.2 Characteristic of GPD2 2
1.3 Characteristic of ether lipids 4
1.4 Objective 5
2. Materials and Methods 6
2.1 Cell preparation 6
2.2 Cell proliferation assay 8
2.3 Clonogenic assay 10
2.4 Wound healing assay 10
2.5 Nuclear magnetic resonance (NMR) spectroscopy 11
2.6 LC-MS analysis 12
2.7 Western blot analysis 15
2.8 GPD2 enzyme activity test 17
2.9 Animal experiment 18
2.10 CCK-8 assay 19
2.11 Respiration measurement 19
2.12 Mitochondrial ATP measurement 20
2.13 ROS measurement 21
2.14 RNA sequencing (RNA-Seq) analysis 22
2.15 DHAP supplementation 23
2.16 Lipid rafts isolation 24
2.17 Cell cycle analysis 24
2.18 Bioinformatics analysis 25
2.19 Statistical analysis 26
3. Results 27
3.1 Mitochondrial damage causes loss of GPD2 activity leading to decreased DHAP/G3P ratio 27
3.1.1 Different phenotype and metabolic profile in ρ0 cells 27
3.1.2 Alteration of DHAP/G3P ratio in ρ0 cell 30
3.1.3 Decreased GPD2 expression and activity in ρ0 cells 33
3.2 GPD2 deletion affects cancer cell growth and tumor progression 37
3.2.1 Effect of GPD2 inhibition on cancer cell growth 37
3.2.2 Changed cellular functions and tumor growth by GPD2 KO 40
3.3 Mitochondrial bioenergetics is not involved in the role of GPD2 in tumor progression 43
3.3.1 Mitochondrial bioenergetics status upon GPD2 KO 43
3.3.2 Mitochondrial activity and ATP production upon GPD2 KO 46
3.3.3 ROS status upon GPD2 KO 50
3.4 GPD2 regulates ether lipid synthesis to control cancer growth 53
3.4.1 Screening of altered metabolic pathways in 4T1 GPD2 KO cells by RNA-Seq 53
3.4.2 Ether lipid biosynthetic pathway 55
3.4.3 Changes in DHAP/G3P ratio upon GPD2 modulation 57
3.4.4 Changes in ether lipid synthetic pathway upon GPD2 KO 60
3.4.5 Difference in lipid species between 4T1 and 4T1 GPD2 KO cells 62
3.4.6 Quantitative level of ether lipid and the effect of plasmalogen supplementation in 4T1 GPD2 KO cells 65
3.4.7 Effect of DHAP supplementation on ether lipid synthesis and cell growth of 4T1 GPD2 KO cells 68
3.5 Ether lipid metabolism is linked to PI3K/Akt pathway 72
3.5.1 Profiling of pathways altered by GPD2 KO by GSEA 72
3.5.2 Different Akt phosphorylation by GPD2 KO 74
3.5.3 Recovery of Akt phosphorylation by DHAP supplementation 76
3.5.4 GPD2-ether lipid-Akt axis in in vivo 79
3.6 Involvement of GPD2 in different types of cancer 82
3.6.1 GPD2 expression and survival in human cancer 82
3.6.2 GPD2 expression and survival in individual human cancer types 85
3.6.3 Effect of GPD2 inhibition on the growth of different types of human cancer cell lines 88
4. Discussion 90
5. Conclusions 98
References 100
Abstract (Korean) 111박
