The role of p62 in the crosstalk between AMPK and NRF2 induced by metabolic stress in the tumor microenvironment and its implications for the development of anticancer strategies
- 주제(키워드) AMPK , NRF2 , p62 , AXIN , Lysosome. NSCLC
- 주제(DDC) 615.1
- 발행기관 아주대학교 일반대학원
- 지도교수 Sun-Young Chang
- 발행년도 2024
- 학위수여년월 2024. 8
- 학위명 박사
- 학과 및 전공 일반대학원 약학과
- 실제URI http://www.dcollection.net/handler/ajou/000000034168
- 본문언어 한국어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
In the context of nutrient deficiency and toxic accumulation, which are characteristics of the tumor microenvironment, tumor cells have efficient adaptation strategies to metabolic stress for survival. In LKB1-mutant NSCLC, co-occurring mutations in KEAP1 activate NRF2 to compensate for the loss of LKB1-AMPK activity during metabolic adaptation. Here, we found that metabolic stress increases the expression and phosphorylation of p62, which is essential for the activation of the LKB1-AMPK pathway and NRF2, a regulator of antioxidant gene expression, thereby enhancing antioxidant defense and tumor growth. Activation of AMPK and NRF2 by p62 induces autophagic degradation of KEAP1 and formation of AXIN- LKB1-AMPK complexes in the lysosomal membrane, respectively. In addition, AMPK activation is required for the expression and phosphorylation of p62 induced by metabolic stress, confirming the existence of a double positive feedback loop between AMPK and p62. AMPK, which is activated by metabolic stress, increases the expression of p62 by dephosphorylation of TFE3/TFEB in a PP2A-dependent manner, and activates NRF2 through KEAP1 degradation. Furthermore, p62 phosphorylation was increased through TAK1 activated by ROS and lysosomal calcium secretion. Importantly, S24 and S226 phosphorylation of p62 are essential for AMPK and NRF2 activation. Taken together, our results demonstrate a novel double positive feedback loop between AMPK and p62, which leads to the simultaneous activation of AMPK and NRF2. AMPK; p62(SQSTM1); NRF2; Lysosome Biogenesis; NSCLC
more목차
Ⅰ. Introduction 1
Ⅱ. Materials and Methods 3
1. Cell culture, Transduction, Reagents 3
2. Western blot 4
3. Co-immunoprecipitation · 4
4. Cell Fraction 5
5. Plasmid Cloning and Lentivirus Production · 5
6. Quantitative PCR 6
7. Immuno staining 7
8. Lysosome pH 7
9. ROS measurement 8
10. Luciferase assay 8
11. Colony forming assay 8
12. Soft agar assay 9
13. Animal study 9
14. Tumor xenograft 10
15. Acknowledgments 10
Ⅲ. Results 13
1. Metabolic and oxidative stresses activate both the LKB 1-AMPK and the KEAP1-NRF2 pathways 13
2. Metabolic stress activates the p62-KEAP1-NRF2 pathway through ROS-dependent and ROS-independent mechanisms 18
3. p62 promotes the activation of AMPK via inducing lysosomal Axin-LKB1-AMPK complex during metabolic stress 21
4. Dual activation of AMPK and NRF2 synergizes antioxidant defense and tumor growth 25
5. LKB1-AMPK-de pendent expression of p62 is required for NRF2 activation during metabolic stress 30
6. AMPK activation during metabolic stress induces p 62 expression via TFE3/TFEB 35
7. AMPK induces p62 expression via PP2A dependent dephosphorylation of TFE3/TFEB during metabolic stress 38
8. AMPK induces deacidification of lysosome during metabolic stress to trigger PP2A-TFEB/TFE3-p62 expression axis 42
9 . ROS dependent ly s os omal Ca 2 + -TAK1-dependent phosphorylation of p62 is required for NRF2 activation during metabolic stress 50
10. Activation of AMPK and NRF2 and phosphorylation of p62(S24/S226) by ROS-TAK1 are important during metabolic stress 57
Ⅳ. Discussion 65
Ⅴ. Reference 67
Ⅵ. 국문초록 72
