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中华细胞与干细胞杂志(电子版) ›› 2024, Vol. 14 ›› Issue (01) : 1 -10. doi: 10.3877/cma.j.issn.2095-1221.2024.01.001

论著

基于转录组测序分析人大细胞肺癌NCI-H460细胞对类泛素化抑制剂MLN4924的潜在耐药机制
邢磊1, 史镜琪2, 李荣艳3, 刘静2, 刘建伟2, 叶玲2, 张明华3, 范皎2,()   
  1. 1. 100853 北京,解放军医学院,解放军总医院第二医学中心老年医学研究所
    2. 100853 北京,中国人民解放军总医院第二医学中心老年医学研究所 国家老年疾病临床医学研究中心
    3. 100853 北京,中国人民解放军总医院医疗保障中心
  • 收稿日期:2023-11-19 出版日期:2024-02-01
  • 通信作者: 范皎
  • 基金资助:
    北京市科技新星计划(20220484020)

Mechanisms of drug resistance to the neddylation inhibitor MLN4924 in NCI-H460 cells based on transcriptome sequencing analysis

Lei Xing1, Jingqi Shi2, Rongyan Li3, Jing Liu2, Jianwei Liu2, Ling Ye2, Minghua Zhang3, Jiao Fan2,()   

  1. 1. Medical school of Chinese PLA, Institute of Geriatrics, the Second Medical Center, Chinese PLA General Hospital, Beijing 100853, China
    2. Institute of Geriatrics, the Second Medical Center, Chinese PLA General Hospital, National Clinical Research Center for Geriatric Diseases, Beijing 100853, China
    3. Medical Supplies Center of PLA General Hospital, Beijing 100853, China
  • Received:2023-11-19 Published:2024-02-01
  • Corresponding author: Jiao Fan
引用本文:

邢磊, 史镜琪, 李荣艳, 刘静, 刘建伟, 叶玲, 张明华, 范皎. 基于转录组测序分析人大细胞肺癌NCI-H460细胞对类泛素化抑制剂MLN4924的潜在耐药机制[J]. 中华细胞与干细胞杂志(电子版), 2024, 14(01): 1-10.

Lei Xing, Jingqi Shi, Rongyan Li, Jing Liu, Jianwei Liu, Ling Ye, Minghua Zhang, Jiao Fan. Mechanisms of drug resistance to the neddylation inhibitor MLN4924 in NCI-H460 cells based on transcriptome sequencing analysis[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2024, 14(01): 1-10.

目的

探讨人大细胞肺癌NCI-H460细胞对类泛素化抑制剂MLN4924潜在新型耐药机制。

方法

利用MLN4924处理NCI-H460细胞,筛选和建立耐药细胞株。对普通NCI-H460对照细胞、MLN4924处理8 h加药细胞株、48 h加药细胞株和建立成功的耐药株进行高通量转录组测序。根据测序结果筛选差异表达基因,并进行通路富集分析。多组间比较采用单因素方差分析,组间两两比较采用Dunnett-t检验。

结果

共构建3个NCI-H460耐药细胞株。转录组测序分析显示,与对照比较,MLN4924处理8 h加药株、处理48 h加药株、三株耐药株分别筛选出5 303、4 186、3 388、3 675和3 267个差异表达基因,包括ABCA9ABCA13CYR61CYP39A1等基因。RT-qRCR实验进一步验证了在瞬时加药组和耐药株中ABCA9CYR61基因高表达。GO富集分析结果显示,差异表达基因与细胞周期、细胞凋亡、细胞间信号转导和细胞黏附等细胞生物过程高度相关,还与细胞核和细胞膜等细胞组分密切相关。KEGG通路富集分析结果显示,耐药细胞中差异表达基因集中在MAPK信号通路、PI3K/Akt信号通路和cAMP信号通路等。

结论

在人大细胞肺癌NCI-H460细胞中,ABCA9CYR61ABCC8CYP4F12等基因的表达变化可能会提高其对MLN4924的耐药能力。这种耐药能力可能与MAPK信号通路、PI3K/Akt信号通路和cAMP信号通路有关。

Objective

This study aims to explore potential novel drug resistance mechanism of NCI-H460 cells to the neddylation inhibitor MLN4924.

Methods

NCI-H460 cells were treated by MLN4924 to screen and establish drug-resistant cell lines. High throughput transcriptome sequencing was performed on the drug-resistant cell lines, MLN4924 treated 8-hour cell lines, 48-hour cell lines, and control cells. Then we screened differentially expressed genes based on sequencing results and conducted pathway enrichment analysis. The comparison among multiple groups was used by one-way ANOVA, and the pairwise comparison between two groups were performed by Dunnett-t test.

Results

Three MLN4924-resistant cell lines were constructed. Transcriptome sequencing analysis showed that compared to the normal control group, there were 5 303, 4 186, 3 388, 3 675, and 3267 differentially expressed genes for MLN4924 treated 8-hour, 48-hour cell lines, and three drug-resistant cell lines, respectively, including genes such as ABCA9, ABCA13, CYR61, CYP39A1, etc. The RT-qPCR experiment further confirmed the significant high expression of ABCA9 and CYR61 in the transient treated cells and drug-resistant cells. The GO enrichment analysis results showed that differentially expressed genes were highly correlated with cellular biological processes such as cell cycle, apoptosis, intercellular signal transduction, and cell adhesion, as well as with cell components such as nucleus and cell membrane. KEGG pathway enrichment analysis showed that differentially expressed genes in drug-resistant cells were concentrated in MAPK signaling pathway, PI3K/Akt signaling pathway and cAMP signaling pathway.

Conclusion

In NCI-H460 human lung cancer cells, changes in the expression of genes such as ABCA9, CYR61, ABCC8, and CYP4F12 may enhance their resistance to MLN4924. This resistance may be related to the MAPK signaling pathway, PI3K Akt signaling pathway, and cAMP signaling pathway.

表1 引物序列信息
图1 光镜下观察不同浓度MLN4924作用96 h的NCI-H460细胞(×100)注:a图为正常生长的细胞;b图为0.78 μmol/L MLN4924作用的细胞;c图为1.56 μmol/L MLN4924作用的细胞;d图为2 μmol/L MLN4924作用的细胞
图2 NCI-H460耐药细胞株NMR1、NMR2、NMR3组和对照组细胞随MLN4924浓度变化细胞存活率曲线
图3 主成分分析图和各组细胞株差异表达基因散点图注:a图为NCI-H460细胞耐药株、瞬时加药组和对照组的主成分分析图;b图为各组细胞株差异表达基因散点图
图4 NCI-H460细胞瞬时加药组和耐药细胞株差异表达基因火山图和韦恩图注:a ~ e图为NCI-H460细胞瞬时加药NMS8h、NMS48h组和耐药细胞株NMR1、NMR2、NMR3组mRNA差异表达基因火山图;f图为NCI-H460细胞瞬时加药组NMS8h、NMS48h和耐药细胞株NMR1、NMR2、NMR3差异表达基因韦恩图
图5 差异表达基因热图注:a图为各组细胞株中与药物转运相关基因表达热图;b图为各组细胞株与药物代谢相关基因表达热图;图中的数值代表与对照组细胞比较,各组细胞株的相应基因表达变化倍数,红色代表与对照组细胞相比高表达的基因,蓝色代表与对照组细胞相比低表达的基因,灰色代表此基因在对应的细胞株中未检测到其表达
图6 各组细胞ABCA9CYR61基因mRNA表达水平注:a图为各组细胞ABCA9基因mRNA表达水平柱状图;b图为各组细胞CYR61基因mRNA表达水平柱状图,与对照比较,aP < 0.05
图7 差异表达基因GO富集分析注:a图为NMS8h组;b图为NMS48h组;c图为NMR1组;d图为NMR2组;e图为NMR3组
图8 差异表达基因KEGG通路富集分析注:a图为NMS8h组;b图为NMS48h组;c图为NMR1组;d图为NMR2组;e图为NMR3组
1
Zhou L, Zhang W, Sun Y, et al. Protein neddylation and its alterations in human cancers for targeted therapy[J]. Cell Signal, 2018, 44:92-102.
2
Ni S, Chen X, Yu Q, et al. Discovery of candesartan cilexetic as a novel neddylation inhibitor for suppressing tumor growth[J]. Eur J Med Chem, 2020,185:111848.
3
Deberardinis RJ, Lum JJ, Hatzivassiliou G, et al. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation[J]. Cell Metab, 2008, 7(1):11-20.
4
Claus S. NEDD8-its role in the regulation of Cullin-RING ligases[J]. Curr Opin Plant Biol, 2018, 45:112-119.
5
Ribet D, Cossart P. Ubiquitin, SUMO, and NEDD8: key targets of bacterial pathogens[J]. Trends Cell Biol, 2018, 28(11):926-940.
6
Zhang ZX, Heng YQ, Cheng W, et al. Reactive oxygen species (ROS)-response nanomedicine through knocking down a novel therapeutic target NEDD8-conjugating enzyme UBC12 (UBE2M) in the treatment of liver cancer[J]. Mater Design, 2021, 204:109648
7
Chew EH, Hagen T. Substrate-mediated regulation of cullin neddylation[J]. J Biol Chem, 2007, 282(23):17032-17040.
8
Zhang XY, Zhang YL, Qiu GH, et al. Hepatic neddylation targets and stabilizes electron transfer flavoproteins to facilitate fatty acid β-oxidation[J]. Proc Natl Acad Sci USA, 2020, 117(5): 2473-2483.
9
Kostrhon S, Prabu JR, Baek K, et al. CUL5-ARIH2 E3-E3 ubiquitin ligase structure reveals cullin-specific NEDD8 activation[J]. Nat Chem Biol, 2021, 17(10):1075-1083.
10
Milhollen MA, Thomas MP, Narayanan U, et al. Treatment-emergent mutations in NAEβ confer resistance to the NEDD8-Activating enzyme inhibitor MLN4924[J]. Cancer Cell, 2012, 21(3): 388-401.
11
Ferdosi SR, Taylor B, Lee M, et al. RETRACTED: PTEN loss drives resistance to the neddylation inhibitor MLN4924 in glioblastoma and can be overcome with TOP2A inhibitors[J]. Neuro Oncol, 2022, 24(11):1857-1868.
12
Kathawala RJ, Espitia CM, Jones TM, et al. ABCG2 overexpression contributes to pevonedistat resistance[J]. Cancers, 2020, 12(2):429.
13
Li Y, Plesescu M, Prakash SR. Synthesis of two isotopically labeled versions of NEDD8-activating enzyme (NAE) inhibitor[J]. Tetrahedron Lett, 2011, 52(15):1807-1810.
14
秦小琰,刘彦尧, 康权. 肝癌肝移植术后复发转移的防治策略和靶向免疫治疗[J]. 器官移植, 2022, 13(2): 271-276.
15
Jiang YY, Cheng W, Li LH, et al. Effective targeting of the ubiquitin-like modifier NEDD8 for lung adenocarcinoma treatment[J]. Cell Biol Toxicol, 202, 36(4):349-364.
16
Pasello M, Giudice AM, Scotlandi K. The ABC subfamily A transporters:multifaceted players with incipient potentialities in cancer[J]. Semin Cancer Biol, 2020, 60:57-71.
17
Smith B, Land H. Anticancer activity of the cholesterol exporter ABCA1 gene[J]. Cell Rep Med, 2012, 2(3):580-590.
18
Liu X, Li Q, Zhou J, et al. ATP-binding cassette transporter A7 accelerates epithelial-to-mesenchymal transition in ovarian cancer cells by upregulating the transforming growth factor-β signaling pathway[J]. Oncol Lett, 2018, 16(5):5868-5874.
19
Erdogan H. One small step for cytochrome P450 in its catalytic cycle, one giant leap for enzymology[J]. J Porphyr Phthalocyanines, 2019, 23(4-5):358-366.
20
Jennifer, Fuentealba M, Cabrera R, et al. Modeling the interfacial interactions between CrtS and CrtR from Xanthophyllomyces dendrorhous, a P450 system involved in astaxanthin production[J]. J Agric Food Chem, 2012, 60(35):8640-8647.
21
Iwama R, Kobayashi S, Ishimaru C, et al. Functional roles and substrate specificities of twelve cytochromes P450 belonging to CYP52 family in n-alkane assimilating yeast Yarrowia lipolytica[J]. Fungal Genet Biol, 2016, 91:43-54.
22
Kelly SL, Kelly DE. Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?[J]. Philos Trans R Soc Lond B Biol Sci, 2013, 368(1612):20120476.
23
Stipp MC, Acco A. Involvement of cytochrome P450 enzymes in inflammation and cancer: a review[J]. Cancer Chemother Pharmacol, 2021, 87(3):295-309.
24
董年,宋晨剑,裘丹萍,等.基质蛋白CCN1与肺部疾病的研究进展[J].中国病理生理杂志,2018,34(1):188-192.
25
Kim EK, Choi EJ. Compromised MAPK signaling in human diseases: an update[J]. Arch Toxikol, 2015, 89(6):867-882.
26
Olea-Flores M, Zuñiga-Eulogio MD, Mendoza-Catalán MA, et al. Extracellular-signal regulated kinase: a central molecule driving epithelial-mesenchymal transition in cancer[J]. Int J Mol Sci, 2019, 20(12):2885.
27
Kiniwa Y, Okuyama R. Recent advances in molecular targeted therapy for unresectable and metastatic BRAF-mutated melanoma[J]. Jpn J Clin Oncol, 2021, 51(3):315-320.
28
Djanani A, Eller S, Fner D, et al. The role of BRAF in metastatic colorectal carcinoma-past, present, and future[J]. Int J Mol Sci, 2020, 21(23):1-21.
29
Yu M, Grady WM. Therapeutic targeting of the phosphatidyl-inositol 3-kinase signaling pathway: novel targeted therapies and advances in the treatment of colorectal cancer[J]. Therap Adv Gastroenterol, 2012, 5(5):319-337.
30
Revathidevi S, Munirajan AK. Akt in cancer: mediator and more[J]. Semin Cancer Biol, 2019, 59:80-91.
31
Nie Y, Sun L, Wu Y, et al. AKT2 regulates pulmonary inflammation and fibrosis via modulating macrophage activation[J]. J Immunol, 2017, 198(11):4470-4480.
32
Mercer PF, Woodcock HV, Eley JD, et al. Exploration of a potent PI3 kinase/mTOR inhibitor as a novel anti-fibrotic agent in IPF[J]. Thorax, 2016, 71(8):701-711.
33
Cheadle C, Nesterova M, Watkins T, et al. Regulatory subunits of PKA define an axis of cellular proliferation/differentiation in ovarian cancer cells[J]. BMC Med Genomics, 2008, 1:43.
34
Casarini L, Santi D, Brigante G, et al. Two hormones for one receptor: evolution, biochemistry, actions, and pathophysiology of LH and hCG[J]. Endocr Rev, 2018, 39(5):549-592.
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