Metabolic Byproducts Suppress Necroptosis to Limit the Efficacy of Chemotherapy = 대사 부산물의 necroptosis 억제를 통한 항암화학요법 효능 저해 기전 연구

다국어 초록 (Multilingual Abstract)

Aberrant cell death mechanisms leading to uncontrolled cell proliferation constitute a hallmark of cancer. While chemotherapy-induced cytotoxicity is crucial for effective cancer treatment, metabolic byproducts frequently impair this process through mechanisms that remain poorly understood. In this study, we demonstrate that acrolein, a byproduct originating from polyamine metabolism, tobacco smoke, or fuel combustion, acts as a dual regulator of cell death: it triggers ferroptosis but simultaneously inhibits necroptosis by blocking the oligomerization of the effector protein MLKL. We found that the depletion of SAT1, an enzyme involved in intracellular polyamine catabolism, renders cancer cells susceptible to necroptosis. Furthermore, mouse tumor models reveal that administering an acrolein-scavenging agent hydralazine overcomes the necroptosis blockade, thereby potentiating the anti-tumor effects of cyclophosphamide. Consistent with these findings, clinical data indicate that cancer patients with elevated necroptosis activity and reduced expression of polyamine catabolizing enzymes show improved survival rates. These results underscore the therapeutic potential of targeting metabolic byproducts to enhance chemotherapeutic efficacy.

국문 초록 (Abstract)

암은 세포 사멸 기전의 이상으로 인해 세포 증식이 적절히 억제되지 못하면서 발생하는 질병이다. 화학요법에 사용되는 약물은 세포 독성을 통해 암을 치료하지만, 그 과정에서 다양한 대사 부산물이 발생한다. 이러한 대사 부산물의 생성은 아직 명확히 규명되지 않은 기전을 통해 세포 사멸 유도를 저해하여 항암 치료 효능을 감소시킨다. 본 연구에서는 폴리아민(polyamine) 대사, 담배 연기, 연료의 연소 등에서 유래하는 대사 부산물인 아크롤레인(acrolein)이 서로 다른 두 세포 사멸 기전에 관여하는 이중 조절자로 작용함을 규명하였다. 아크롤레인은 ferroptosis를 유발하는 동시에, necroptosis를 유도하는 단백질인 MLKL의 올리고머화(oligomerization)을 차단하여 세포 사멸을 억제한다. 이에 더하여, 폴리아민 분해 효소인 SAT1의 결핍은 암세포를 necroptosis에 보다 취약하게 만든다는 사실을 발견하였다. 마우스 종양 모델을 통해, 아크롤레인을 제거하는 약물인 하이드랄라진(hydralazine)을 투여함으로써 necroptosis 억제 현상을 극복하여 사이클로포스파미드(cyclophosphamide)의 항종양 효과를 강화할 수 있음을 확인하였다. 임상 데이터 분석에서, necroptosis 활성이 높으면서 폴리아민 대사 유전자 발현이 낮은 암 환자가 더 높은 생존율을 보이는 것으로 나타났다. 본 연구 결과는 대사 부산물을 표적으로 하는 접근법이 항암 화학요법의 치료 효능을 증대시킬 수 있는 잠재적 전략임을 시사한다.

목차 (Table of Contents)

• Abstract i
• Table of Contents iii
• List of Tables vii
• List of Figures vii
• Chapter 1. Introduction 1
• 1.1. Study background 1
• 1.1.1. Cell death 1
• 1.1.2. Acrolein 4
• 1.1.3. Drug metabolism and cancer 5
• 1.2. Purpose of research 7
• Chapter 2. Methods 8
• 2.1. Animals 8
• 2.2. Generation of immortalized BMDMs 9
• 2.3. Cell culture 9
• 2.4. Cell stimulation 11
• 2.5. Generation of knockout cells 12
• 2.6. Lipid peroxidation quantification 13
• 2.7. Real-time imaging for cell death 13
• 2.8. Immunoblot analysis 14
• 2.9. Quantification of H2O2 levels 16
• 2.10. RNA dot blot analysis 16
• 2.11. Chemoproteomic profiling 17
• 2.12. Non-reducing SDS-PAGE 19
• 2.13. Immunofluorescence staining 20
• 2.14. In vivo experiments 21
• 2.15. Survival analysis 22
• 2.16. Meta-analysis of relative risk estimation 24
• 2.17. Quantification and statistical analysis 24
• Chapter 3. Results 26
• 3.1. Spermine induces cell death via ferroptosis 26
• 3.1.1. Spermine triggers cell death in both mouse and human cells 26
• 3.1.2. Spermine initiates activation of necroptotic cell death molecules 30
• 3.1.3. Spermine activates lipid peroxidation and induces ferroptosis 35
• 3.2. Metabolic byproduct acrolein drives spermine-induced ferroptosis 42
• 3.2.1. Treatment of spermidine, but not putrescine, triggers cell death 42
• 3.2.2. Acrolein scavenger hydralazine abrogates spermine-induced cell death 44
• 3.2.3. High dose of H2O2 causes non-ferroptotic cell death 47
• 3.3. ZBP1, inflammasomes and type I IFN signaling are dispensable for cell death triggered by acrolein 52
• 3.3.1. ZBP1 is not required for acrolein-induced cell death 52
• 3.3.2. Inflammasomes and type I IFN signaling do not contribute to acrolein-induced cell death 54
• 3.4. Acrolein-mediates protein modifications drive ferroptotic cell death 56
• 3.4.1. Chemoproteomic profiling of acrolein-treated cells identifies ferroptosis-associated proteins 56
• 3.4.2. Disruption of iron homeostasis and redox regulation by acrolein contributes to ferroptosis 63
• 3.5. Acrolein impairs MLKL oligomerization and suppresses execution of necroptosis 65
• 3.5.1. Treatment of acrolein inhibits necroptosis induced by TSZ 65
• 3.5.2. Loss of SAT1 increases susceptibility to necroptosis 71
• 3.6. Sequestration of acrolein reinstates necroptosis and enhances the efficacy of anticancer therapy 73
• 3.6.1. The acrolein scavenger hydralazine potentiates cell death in mouse colon cancer cells 73
• 3.6.2. Co-administration of cyclophosphamide and hydralazine augments chemotherapeutic efficacy in mouse tumor models 75
• 3.7. Polyamine metabolism and necroptosis correlate with improved survival of humans with cancer 78
• 3.7.1. Reduced expression of polyamine-catabolizing enzymes associates with improved survival 78
• 3.7.2. Increased mortality among smokers highlights the role of acrolein exposure in chemotherapy response 80
• Chapter 4. Discussion 83
• Data Availability 87
• References 88
• Acknowledgements 95
• Abstract in Korean 96