Inhibitory effects of temozolomide on glioma cells is sensitized by RSL3-induced ferroptosis but negatively correlated with expression of ferritin heavy chain 1 and ferritin light chain

1. 논문 제목: Inhibitory effects of temozolomide on glioma cells is sensitized by RSL3-induced ferroptosis but negatively correlated with expression of ferritin heavy chain 1 and ferritin light chain

2. 저자명: Fei-Cheng Yang, Chuan Wang, Jiang Zhu, Qu-Jing Gai, Min Mao, Jiang He, Yan Qin, Xiao-Xue Yao, Yan-Xia Wang, Hui-Min Lu, Mian-Fu Cao, Ming-Min He, Xian-Mei Wen, Ping Leng, Xiong-Wei Cai, Xiao-Hong Yao, Xiu-Wu Bian and Yan Wang

3. 출처 및 게재 (발행일): Laboratory Investigation, 25 February 2022

4. DOI: https://doi.org/10.1038/s41374-022-00779-7

5. Abstract: Invasive growth of glioblastoma makes residual tumor unremovable by surgery and leads to disease relapse. Temozolomide is widely used first-line chemotherapy drug to treat glioma patients, but development of temozolomide resistance is almost inevitable. Ferroptosis, an iron-dependent form of non-apoptotic cell death, is found to be related to temozolomide response of gliomas. However, whether inducing ferroptosis could affect invasive growth of glioblastoma cells and which ferroptosis-related regulators were involved in temozolomide resistance are still unclear. In this study, we treated glioblastoma cells with RSL3, a ferroptosis inducer, in vitro (cell lines) and in vivo (subcutaneous and orthotopic animal models). The treated glioblastoma cells with wild-type or mutant IDH1 were subjected to RNA sequencing for transcriptomic profiling. We then analyze data from our RNA sequencing and public TCGA glioma database to identify ferroptosis-related biomarkers for prediction of prognosis and temozolomide resistance in gliomas. Analysis of transcriptome data from RSL3-treated glioblastoma cells suggested that RSL3 could inhibit glioblastoma cell growth and suppress expression of genes involved in cell cycle. RSL3 effectively reduced mobility of glioblastoma cells through downregulation of critical genes involved in epithelial-mesenchymal transition. Moreover, RSL3 in combination with temozolomide showed suppressive efficacy on glioblastoma cell growth, providing a promising therapeutic strategy for glioblastoma treatment. Although temozolomide attenuated invasion of glioblastoma cells with mutant IDH1 more than those with wild-type IDH1, the combination of RSL3 and temozolomide similarly impaired invasive ability of glioblastoma cells in spite of IDH1 status. Finally, we noticed that both ferritin heavy chain 1 and ferritin light chain predicted unfavorable prognosis of glioma patients and were significantly correlated with mRNA levels of methylguanine methyltransferase as well as temozolomide resistance. Altogether, our study provided rationale for combination of RSL3 with temozolomide to suppress glioblastoma cells and revealed ferritin heavy chain 1 and ferritin light chain as biomarkers to predict prognosis and temozolomide resistance of glioma patients.

6. 한글 초록 요약본: Temozolomide (TMZ)는 교모세포종의 표준 항암제이지만 대부분의 환자에서 내성이 발생함. 본 연구에서는 GPX4 inhibitor인 RSL3를 이용하여 ferroptosis를 유도하면 TMZ의 항암효과를 높일 수 있는지 평가함. RSL3는 GBM 세포의 증식과 침윤을 억제하고 EMT 관련 유전자 발현을 감소시켰으며 TMZ와 병용 시 세포 및 동물모델 모두에서 항암 효과가 크게 증가함. 또한 FTH1과 FTL이 TMZ 내성 및 MGMT 발현과 양의 상관관계를 보이며 높은 발현은 불량한 예후와 관련됨을 확인함. 따라서 RSL3+TMZ 병용요법은 GBM 치료 전략으로 가능성이 있으며 FTH1/FTL은 TMZ 내성을 예측하는 바이오마커가 될 수 있음을 제시함.

7. 한글 논문 요약본 (Intro, Methods, Results)

7-1) Introduction: 교모세포종(GBM)은 높은 침윤성과 TMZ 내성 때문에 예후가 매우 불량함. Ferroptosis는 GPX4 억제를 통해 유도되는 철 의존적 세포사멸로 최근 TMZ 감수성과 관련이 보고되었지만 GPX4 inhibitor인 RSL3의 효과와 기전은 충분히 연구되지 않았음. 또한 ferroptosis 관련 인자 중 TMZ 내성과 예후를 예측할 수 있는 바이오마커도 부족함. 이에 저자들은 RSL3의 항암효과와 TMZ 병용효과를 평가하고 ferroptosis 관련 바이오마커를 발굴하고자 함.  

7-2) Methods:

-  세포 실험: LN18, LN229, GBM1, IDH1 WT 및 IDH1 R132H mutant GBM cell

- 동물 실험: Subcutaneous 및 orthotopic GBM mouse model

-처리군: Vehicle, RSL3, TMZ, RSL3+TMZ

-분석: Cell viability (CCK-8), Wound healing, Transwell invasion, TEM, RNA-seq, Western blot, IHC, TCGA 분석 (FTH1/FTL, MGMT 상관성)

7-3) Results

7-3-1) RSL3는 GBM의 증식과 침윤을 억제함; RSL3는 GBM 세포의 생존율을 감소시키고 migration 및 invasion을 억제함. RNA-seq에서는 cell cycle과 EMT 관련 유전자가 감소하였으며 EMT marker (N-cadherin, Vimentin, Snail, Slug, Twist1) 발현도 감소함. 이는 apoptosis가 아닌 ferroptosis에 의한 효과였음.

7-3-2) RSL3는 TMZ 감수성을 증가시킴; RSL3를 병용하면 TMZ의 IC50이 크게 감소하여 TMZ 감수성이 증가함. 또한 invasion 억제 효과가 단독 처리보다 우수했으며 TEM에서는 RSL3와 TMZ 병용 시 ferroptosis의 특징인 미토콘드리아 손상이 가장 심하게 나타남.

7-3-3) In vivo에서도 병용효과를 확인함; Subcutaneous 및 orthotopic GBM 모델에서 RSL3와 TMZ 병용군은 가장 강한 종양 성장 억제 효과를 보임. Ki67 발현이 감소하였고 GPX4도 억제되었으며 TEM에서도 ferroptosis가 확인됨.  

7- 3-4) IDH1 상태와 관계없이 RSL3가 효과가 보임; IDH1 mutant 세포는 RSL3에 더 민감했으며 TMZ 단독 효과도 더 좋았음. 그러나 RSL3+TMZ 병용 시에는 IDH1 WT와 mutant 모두에서 침윤 억제 효과가 크게 증가하여 병용 효과는 IDH1 상태와 관계없이 나타남.

7-3-5) FTH1과 FTL은 TMZ 내성과 밀접하게 관련됨; TCGA 분석에서는 FTH1과 FTL은 GPX4 및 MGMT와 가장 높은 상관관계를 보임. 두 유전자의 발현이 높을수록 EMT, mesenchymal subtype, TMS resistance gene signature가 증가함. 또한 FTH1 또는 FTL을 knockdown하면 TMZ 감수성이 증가함.

7-3-6) FTH1과 FTL은 예후 예측 바이오마커임; Glioma 조직과 TCGA 데이터를 분석한 결과, FTH1과 FTL 발현은 grade가 증가할수록 높아졌으며 높은 발현은 overall survival과 progression-free survival 감소와 연관됨. 특히 FTH1High/FTLHigh 환자군에서 가장 불량한 예후를 보임. 또한 mesenchymal subtype에서 높은 발현을 나타냄.

8. 대표 figure

Fig 3. Inhibition of GBM cell growth in vivo by RSL3 and TMZ.