NF2/YAP信号通路通过FSP1诱导CD24高表达的三阴性乳腺癌细胞铁死亡

doi:10.3877/cma.j.issn.1674-0807.2024.04.003

中华乳腺病杂志(电子版) ›› 2024, Vol. 18 ›› Issue (04) : 206 -211. doi: 10.3877/cma.j.issn.1674-0807.2024.04.003

论著

NF2/YAP信号通路通过FSP1诱导CD24高表达的三阴性乳腺癌细胞铁死亡

蒲卢兰¹, 李静佳², 陈宇¹, 周瑜清¹, 荣欣欣¹, 侯令密², 周方方¹^,^†(

)

^1. 637000　四川南充，川北医学院基础医学与法医学院
^2. 637000　四川南充，川北医学院附属医院甲状腺乳腺外科/乳腺癌生物靶向实验室

收稿日期:2023-11-14 出版日期:2024-08-01
通信作者: 周方方
基金资助:
四川省科技厅自然科学基金面上项目(2022NSFSC0775); 四川省基层卫生事业发展研究中心资助项目(SWFZ22-C-82); 南充市科技局市校科技战略合作项目(20SXQT0052); 川北医学院校级科研发展计划项目(CBY22-QNA11)

NF2/YAP signaling pathway induces ferroptosis in triple negative breast cancer cells with high expression of CD24 by FSP1

Lulan Pu¹, Jingjia Li², Yu Chen¹, Yuqing Zhou¹, Xinxin Rong¹, Lingmi Hou², Fangfang Zhou¹^,^†()

^1. School of Basic Medicine and Forensic Medicine, North Sichuan Medical College, Nanchong 637000, China
^2. Department of Breast and Thyroid Surgery, Biological Targeting Laboratory of Breast Cancer, Affiliated Hospital of North Sichuan Medical College, Nanchong 637000, China

Received:2023-11-14 Published:2024-08-01
Corresponding author: Fangfang Zhou

全文 ( ) 下载PDF ( ) 导出引用

引用本文：

蒲卢兰, 李静佳, 陈宇, 周瑜清, 荣欣欣, 侯令密, 周方方. NF2/YAP信号通路通过FSP1诱导CD24高表达的三阴性乳腺癌细胞铁死亡[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(04): 206-211.

Lulan Pu, Jingjia Li, Yu Chen, Yuqing Zhou, Xinxin Rong, Lingmi Hou, Fangfang Zhou. NF2/YAP signaling pathway induces ferroptosis in triple negative breast cancer cells with high expression of CD24 by FSP1[J/OL]. Chinese Journal of Breast Disease(Electronic Edition), 2024, 18(04): 206-211.

目的

研究NF2和YAP表达对CD24高表达的三阴性乳腺癌细胞铁死亡的影响，探讨铁死亡的相关分子机制。

方法

培养CD24^high和CD24^lowMDA-MB-231细胞，通过台盼蓝染色(TBA)实验检测经半胱氨酸饥饿、铁死亡诱导剂Erastin和RSL3处理后细胞的死亡情况，TBA法检测丙二醛(MDA)含量，分析细胞铁死亡水平。使用Western blot检测NF2和YAP在CD24^high和CD24^lowMDA-MB-231细胞中的蛋白表达情况，并用慢病毒分别进行敲除和过表达，观察细胞铁死亡水平，并采用实时定量RT-PCR和Western blot检测成纤维细胞特异蛋白1(FSP1)的表达变化。

结果

经半胱氨酸饥饿、铁死亡诱导剂Erastin和RSL3分别处理后MDA-MB-231细胞死亡率增加，CD24^low细胞死亡率高于CD24^high细胞(t＝14.548，P<0.001；t＝8.310，P＝0.001；t＝8.600，P＝0.001)。同时，TBA法结果显示，在含或不含半胱氨酸条件下，CD24^high和CD24^lowMDA-MB-231细胞的MDA含量差异有统计学意义(t＝－3.920，P＝0.017；t＝11.566，P<0.001)；与对照组(DMSO)相比，CD24^high和CD24^low细胞经铁死亡诱导剂Erastin和RSL3处理后MDA水平增加，其中CD24^low细胞的MDA含量比CD24^high更高(t＝10.763，P＝0.006；t＝24.067，P<0.001)。Western blot实验结果表明，NF2与YAP在CD24^low和CD24^high细胞的蛋白含量差异有统计学意义(t＝－4.331，P＝0.012；t＝4.219，P＝0.013)，CD24^low细胞中YAP的蛋白表达更高(t＝4.219，P＝0.013)，而CD24^high细胞中NF2的蛋白表达更高(t＝－4.331，P＝0.012)。在CD24^highMDA-MB-231细胞中构建NF2敲除或(和)YAP过表达细胞株，并用实时定量RT-PCR和Western blot检测，结果显示铁死亡关键蛋白FSP1在4组间的表达差异有统计学意义(F＝30.297，P<0.001)。同时，检测NF2敲除或(和)YAP过表达后CD24^highMDA-MB-231细胞的铁死亡情况，结果表明敲除NF2或(和)过表达YAP后加入铁死亡诱导剂Erastin均可在一定程度上促进细胞死亡(F＝38.911，P<0.050)，其中在NF2敲除联合YAP过表达条件下，CD24^highMDA-MB-231细胞死亡率最高，而在Erastin的基础上添加铁死亡抑制剂Fer-1后细胞的死亡率下降，4组间比较差异无统计学意义(F＝0.256，P＝0.855)。

结论

在CD24高表达的三阴性乳腺癌细胞中敲除NF2联合过表达YAP会抑制FSP1表达，促进细胞铁死亡。

关键词: 乳腺肿瘤, 铁死亡, 成纤维细胞特异蛋白1, NF2, YAP

Objective

To observe the impact of NF2 and YAP expression on ferroptosis of triple negative breast cancer cells with high expression of CD24 and explore the related molecular mechanism.

Methods

MDA-MB-231 breast cancer cells with high expression of CD24 (CD24^high) and low expression of CD24 (CD24^low) were cultured. The cells were treated with ferroptosis agonists Erastin, RSL3 and cysteine starvation. The cell death was detected by Trypan blue staining (TBA). Malondialdehyde(MDA)content was measured using the TBA method to analyze the level of cellular ferroptosis. Western blot analysis was used to detect the protein expression of NF2 and YAP in CD24^high and CD24^low MDA-MB-231 cells. Lentiviral vectors were used to knock out and over-express NF2 and YAP, and changes in cellular ferroptosis were observed. The real-time quantitative RT-PCR and Western blot analysis were used to detect the expression of fibroblast-specific protein 1(FSP1)after NF2 knock out and YAP over-expression.

Results

After treatment with cysteine starvation, the ferroptosis inducers Erastin and RSL3, the death rate of MDA-MB-231 cells increased, with the death rate of CD24^low cells significantly higher than that of CD24^high cells (t=14.548, P<0.001; t=8.310, P=0.001; t=8.600, P=0.001). Meanwhile, the TBA method results showed that there was a significant difference in MDA content between CD24^high and CD24^low MDA-MB-231 cells under conditions with or without cysteine (t=-3.920, P=0.017; t=11.566, P<0.001); compared with the control group with DMSO, the MDA levels of CD24^high and CD24^low cells significantly increased after treatment with the ferroptosis inducers Erastin and RSL3, with the MDA content of CD24^low cells significantly higher than that of CD24^high cells (t=10.763, P=0.006; t=24.067, P<0.001). Western blot experiments showed that there was a significant difference in the protein content of NF2 and YAP between CD24^low and CD24^high MDA-MB-231 cells (t=-4.331, P=0.012; t=4.219, P=0.013), with higher expression of YAP protein in CD24^low cells (t=4.219, P=0.013) and higher expression of NF2 protein in CD24^high cells (t=-4.331, P=0.012). NF2 knockout or (and) YAP overexpression cell lines were constructed in CD24^high cells, and real-time quantitative RT-PCR and Western blot analysis were used to detect the expression of the ferroptosis key protein FSP1 among the four groups (F=30.297, P<0.001). Meanwhile, the ferroptosis situation of CD24^high MDA-MB-231 cells after NF2 knockout or (and) YAP overexpression was detected, and the results showed that knocking out NF2 or (and) overexpressing YAP could promote cell death to a certain extent after adding the ferroptosis inducer Erastin (F=38.911, P<0.050), with the highest death rate of CD24^high MDA-MB-231 cells under the condition of NF2 knockout combined with YAP overexpression. The addition of the ferroptosis inhibitor Fer-1 after Erastin led to a decrease in the death rate of cells, with no statistically significant difference among the four groups (F=0.256, P=0.855).

Conclusions

Knocking out NF2 and overexpressing YAP in CD24^high triple negative breast cancer cells can inhibit the expression of FSP1 and promote cell ferroptosis.

Key words: Breast neoplasms, Ferroptosis, Fibroblast-specific protein 1, NF2, YAP

表1 实时荧光定量反转录PCR引物序列

引物名称	引物序列(5’→3’)
NF2	正向：GTGCCATACTAAGCAGCTTGC
NF2	反向：ACACTACAGCTCGGAAATCCA
YAP	正向：GTCGGAACCCTTCCCTCATTA
YAP	反向：GGCCTTCACTGACAGGTAGTA
FSP1	正向：ATCGTCCGAGATGATGTTGATGGG
FSP1	反向：ATTACTGCTACTCCGTACACCACTG
GAPDH	正向：TGACTTCAACAGCGACACCCA
GAPDH	反向：CACCCTGTTGCTGTAGCCAAA

表1 实时荧光定量反转录PCR引物序列

引物名称	引物序列(5’→3’)
NF2	正向：GTGCCATACTAAGCAGCTTGC
NF2	反向：ACACTACAGCTCGGAAATCCA
YAP	正向：GTCGGAACCCTTCCCTCATTA
YAP	反向：GGCCTTCACTGACAGGTAGTA
FSP1	正向：ATCGTCCGAGATGATGTTGATGGG
FSP1	反向：ATTACTGCTACTCCGTACACCACTG
GAPDH	正向：TGACTTCAACAGCGACACCCA
GAPDH	反向：CACCCTGTTGCTGTAGCCAAA

表2 半胱氨酸饥饿处理后CD24^low与CD24^high细胞死亡率比较

组别	实验次数	含半胱氨酸	半胱氨酸饥饿
CD24^low	3	4.60±0.63	28.93±0.40
CD24^high	3	3.83±0.81	12.60±1.82
t值		1.301	14.548
P值		0.263	<0.001

表2 半胱氨酸饥饿处理后CD24^low与CD24^high细胞死亡率比较

组别	实验次数	含半胱氨酸	半胱氨酸饥饿
CD24^low	3	4.60±0.63	28.93±0.40
CD24^high	3	3.83±0.81	12.60±1.82
t值		1.301	14.548
P值		0.263	<0.001

表3 铁死亡诱导剂处理CD24^low与CD24^high细胞死亡率比较

组别	实验次数	DMSO	Erastin	RSL3
CD24^low	3	4.93±1.36	38.15±2.92	39.51±2.16
CD24^high	3	3.61±1.12	14.73±3.91	18.90±1.79
t值		1.292	8.310	8.600
P值		0.266	0.001	0.001

表3 铁死亡诱导剂处理CD24^low与CD24^high细胞死亡率比较

组别	实验次数	DMSO	Erastin	RSL3
CD24^low	3	4.93±1.36	38.15±2.92	39.51±2.16
CD24^high	3	3.61±1.12	14.73±3.91	18.90±1.79
t值		1.292	8.310	8.600
P值		0.266	0.001	0.001

表4 半胱氨酸饥饿处理后CD24^low与CD24^high细胞MDA水平比较

组别	实验次数	含半胱氨酸	半胱氨酸饥饿
CD24^low	3	0.32±0.02	1.63±0.06
CD24^high	3	0.37±0.01	0.96±0.08
t值		-3.920	11.566
P值		0.017	<0.001

表4 半胱氨酸饥饿处理后CD24^low与CD24^high细胞MDA水平比较

组别	实验次数	含半胱氨酸	半胱氨酸饥饿
CD24^low	3	0.32±0.02	1.63±0.06
CD24^high	3	0.37±0.01	0.96±0.08
t值		-3.920	11.566
P值		0.017	<0.001

表5 铁死亡诱导剂处理后CD24^low与CD24^high细胞MDA水平比较

组别	实验次数	DMSO	Erastin	RSL3
CD24^low	3	0.42±0.04	1.76±0.02	1.67±0.02
CD24^high	3	0.40±0.03	1.28±0.08	1.22±0.03
t值		0.970	10.763	24.067
P值		0.387	0.006	<0.001

表5 铁死亡诱导剂处理后CD24^low与CD24^high细胞MDA水平比较

组别	实验次数	DMSO	Erastin	RSL3
CD24^low	3	0.42±0.04	1.76±0.02	1.67±0.02
CD24^high	3	0.40±0.03	1.28±0.08	1.22±0.03
t值		0.970	10.763	24.067
P值		0.387	0.006	<0.001

图1 Western blot检测CD24^low和CD24^highMDA-MB-231细胞中NF2和YAP蛋白表达情况

图2 NF2敲除联合YAP过表达降低CD24^highMDA-MB-231细胞中FSP1表达注：a代表对照组；b代表YAP过表达组(YAP-OV)；c代表NF2敲除组(NF2-KO)；d代表NF2敲除联合YAP过表达组(YAP-OV＋NF2-KO)

表6 NF敲除联合YAP过表达CD24^high细胞加药处理后死亡率比较

组别	实验次数	DMSO	Erastin	Erastin＋Fer-1
对照	3	5.74±0.50	19.89±0.68	6.37±2.50
YAP-OV	3	6.64±1.33	29.43±0.53	6.68±0.78
NF2-KO	3	5.86±1.34	36.31±6.10	7.42±1.48
YAP-OV＋NF2-KO	3	6.19±0.60	46.28±0.57	7.01±0.71
F值		0.469	38.911	0.256
P值		0.712	<0.001	0.855

表6 NF敲除联合YAP过表达CD24^high细胞加药处理后死亡率比较

组别	实验次数	DMSO	Erastin	Erastin＋Fer-1
对照	3	5.74±0.50	19.89±0.68	6.37±2.50
YAP-OV	3	6.64±1.33	29.43±0.53	6.68±0.78
NF2-KO	3	5.86±1.34	36.31±6.10	7.42±1.48
YAP-OV＋NF2-KO	3	6.19±0.60	46.28±0.57	7.01±0.71
F值		0.469	38.911	0.256
P值		0.712	<0.001	0.855

[1]	Franzoi MA, Romano E, Piccart M. Immunotherapy for early breast cancer: too soon, too superficial, or just right？[J].Ann Oncol, 2021, 32(3)：323-336.
[2]	Waks AG, Winer EP. Breast cancer treatment[J]. JAMA, 2019, 321(3)：316.
[3]	Stockwell BR, Jiang X, Gu W. Emerging mechanisms and disease relevance of ferroptosis[J]. Trends Cel Biol, 2020, 30(6)：478-490.
[4]	Mou Y, Wang J, Wu J, et al. Ferroptosis, a new form of cell death: opportunities and challenges in cancer[J]. J Hematol Oncol, 2019, 12(1)：34.
[5]	Hou L, Pu L, Chen Y, et al. Targeted intervention of NF2-YAP signaling axis in CD24-overexpressing cells contributes to encouraging therapeutic effects in TNBC[J]. ACS Nano, 2022, 16(4)：5807-5819.
[6]	Barkal AA, Brewer R, Markovic M, et al. CD24 signalling through macrophage siglec-10 is a target for cancer immunotherapy [J]. Nature, 2019, 572(7769)：392-396.
[7]	Pu L, Wang D, Zhou Y, et al. Expression of CD24 in triple negative breast cancer and its relationship with EMT[J]. Modern Oncol, 2023, 31(2)：217-221.
[8]	Li L, Gong Y, Tang J, et al. ZBTB28 inhibits breast cancer by activating IFNAR and dual blocking CD24 and CD47 to enhance macrophages phagocytosis[J]. Cell Mol Life Sci, 2022, 79(2)：83.
[9]	Stockwell BR, Jiang X, Gu W. Emerging mechanisms and disease relevance of ferroptosiss[J]. Trends Cell Biol, 2020, 30(6)：478-490.
[10]	Guo Y, Chen G. Research progress on iron death regulatory mechanism and tumor occurrence and treatmens[J]. Adv Physiol Sci, 2019, 50(2)：88-93.
[11]	Liang C, Zhang X, Yang M, et al. Recent progress in ferroptosis inducers for cancer therapys[J]. Adv Mater, 2019, 31(51)：e1904197.
[12]	Xie X, Fan T, Zhang B, et al. Research progress on the correlation between iron death and breast cancer treatments[J]. Chin J Clin Oncol, 2020, 47(8)：423-426.
[13]	Doll S, Freitas FP, Shah R, et al. FSP1 is a glutathione-independent ferroptosis suppressors[J]. Nature, 2019, 575(7784)：693-698.
[14]	Bersuker K, Hendricks JM, Li Z, et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosiss[J]. Nature, 2019, 575(7784)：688-692.
[15]	Kosciuk T, Lin H. N-Myristoyltransferase as a glycine and lysine myristoyltransferase in cancer, immunity, and infectionss[J]. ACS Chem Biol, 2020, 15(7)：1747-1758.
[16]	Xu F, Chen WD, Guo MH, et al. Progress of ferroptosis mechanism and application in lung cancer therapys[J]. J Basic clinMed, 2021, 41：442.
[17]	Wu J, Minikes AM, Gao M, et al. Intercellular interaction dictates cancer cell ferroptosis via NF2-YAP signallings[J]. Nature, 2019, 572(7769)：402-406.
[18]	Coates JT, Sun S, Leshchiner I, et al. Parallel genomic alterations of antigen and payload targets mediate polyclonal acquired clinical resistance to sacituzumab govitecan in triple-negative breast cances[J]. Cancer Discov, 2021, 11(10)：2436-2445.
[19]	Gilad N, Zukerman H, Pick M, et al. The role of CD24 in multiple myeloma tumorigenicity and effects of the microenvironment on its expressions[J]. Oncotarget, 2019, 10(52)：5480-5491.
[20]	Ni YH, Zhao X, Wang W. CD24, a review of its role in tumor diagnosis, progression and therapys[J]. Curr Gene Ther, 2020, 20(2)：109-126.
[21]	Dong F． Meta-analysis of the expression of CD24 in breast cancer and its correlation with clinical pathological characteristics and prognosis[D]. Jilin University, 2020, 1-35.
[22]	Doll S, Freitas FP, Shah R, et al. FSP1 is a glutathione-independent ferroptosis suppressors[J]. Nature, 2019, 575(7784)：693-698.
[23]	Meng Z, Moroishi T, Guan KL. Mechanisms of hippo pathway regulations[J]. Genes Dev, 2016, 30(1)：1-17.
[24]	Huang LS, Sudhadevi T, Fu P, et al. Sphingosine kinase 1/S1P signaling contributes to pulmonary fibrosis by activating Hippo/YAP pathway and mitochondrial reactive oxygen species in lung fibroblastss[J]. Int J Mol Sci, 2020, 21(6)：2064.
[25]	Liu-Chittenden Y, Huang B, Shim JS, et al. Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAPs[J]. Genes Dev, 2012, 26(12)：1300-1305.
[26]	Zhang R, Gao Y, Zhao X, et al. FSP1-positive fibroblasts are adipogenic niche and regulate adipose homeostasiss[J]. PLoS Biol, 2018, 16(8)：e2001493.
[27]	Wang D, Dai C, Li Y, et al. Canonical Wnt/β-catenin signaling mediates transforming growth factor-β1-driven podocyte injury and proteinurias[J]. Kidney Int, 2011, 80(11)：1159-1169.
[28]	Mou Y, Wang J, Wu J, et al. Ferroptosis, a new form of cell death: opportunities and challenges in cancers[J]. J Hematol Oncol, 2019, 12(1)：34.

[1]	李洋, 蔡金玉, 党晓智, 常婉英, 巨艳, 高毅, 宋宏萍. 基于深度学习的乳腺超声应变弹性图像生成模型的应用研究[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(06): 563-570.
[2]	周荷妹, 金杰, 叶建东, 夏之一, 王进进, 丁宁. 罕见成人肋骨郎格汉斯细胞组织细胞增生症被误诊为乳腺癌术后骨转移一例[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 380-383.
[3]	河北省抗癌协会乳腺癌专业委员会护理协作组. 乳腺癌中心静脉通路护理管理专家共识[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 321-329.
[4]	刘晨鹭, 刘洁, 张帆, 严彩英, 陈倩, 陈双庆. 增强MRI 影像组学特征生境分析在预测乳腺癌HER-2 表达状态中的应用[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 339-345.
[5]	张晓宇, 殷雨来, 张银旭. 阿帕替尼联合新辅助化疗对三阴性乳腺癌的疗效及预后分析[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 346-352.
[6]	邱琳, 刘锦辉, 组木热提·吐尔洪, 马悦心, 冷晓玲. 超声影像组学对致密型乳腺背景中非肿块型乳腺癌的诊断价值[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 353-360.
[7]	程燕妮, 樊菁, 肖瑶, 舒瑞, 明昊, 党晓智, 宋宏萍. 乳腺组织定位标记夹的应用与进展[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 361-365.
[8]	涂盛楠, 胡芬, 张娟, 蔡海峰, 杨俊泉. 天然植物提取物在乳腺癌治疗中的应用[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 366-370.
[9]	朱文婷, 顾鹏, 孙星. 非酒精性脂肪性肝病对乳腺癌发生发展及治疗的影响[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(06): 371-375.
[10]	葛睿, 陈飞, 李杰, 李娟娟, 陈涵. 多基因检测在早期乳腺癌辅助治疗中的应用价值[J/OL]. 中华乳腺病杂志(电子版), 2024, 18(05): 257-263.
[11]	高杰红, 黎平平, 齐婧, 代引海. ETFA和CD34在乳腺癌中的表达及与临床病理参数和预后的关系研究[J/OL]. 中华普外科手术学杂志(电子版), 2025, 19(01): 64-67.
[12]	韩萌萌, 冯雪园, 马宁. 乳腺癌改良根治术后桡神经损伤1例[J/OL]. 中华普外科手术学杂志(电子版), 2025, 19(01): 117-118.
[13]	张志兆, 王睿, 郜苹苹, 王成方, 王成, 齐晓伟. DNMT3B与乳腺癌预后的关系及其生物学机制[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 624-629.
[14]	王玲艳, 高春晖, 冯雪园, 崔鑫淼, 刘欢, 赵文明, 张金库. 循环肿瘤细胞在乳腺癌新辅助及术后辅助治疗中的应用[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 630-633.
[15]	赵林娟, 吕婕, 王文胜, 马德茂, 侯涛. 超声引导下染色剂标记切缘的梭柱型和圆柱型保乳区段切除术的效果研究[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 634-637.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

NF2/YAP signaling pathway induces ferroptosis in triple negative breast cancer cells with high expression of CD24 by FSP1

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

NF2/YAP signaling pathway induces ferroptosis in triple negative breast cancer cells with high expression of CD24 by FSP1