Shang Fangjian, Nie Hongfeng, Du Liyan, Shang Jin, Song Xiangquan, Chen Ya, Li Hui, Wang Zhuo, Qi Yixin, Zhao Liyan
Department of General Surgery, The First Hospital of Hebei Medical University, Shijiazhuang, 050023, Hebei, China.
Department of Gland Surgery, Hebei General Hospital, Shijiazhuang, 050051, Hebei, China.
Discov Oncol. 2025 May 8;16(1):687. doi: 10.1007/s12672-025-02470-x.
Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer characterized by a high recurrence rate and a lack of effective targeted therapies. The purpose of this study was to investigate the interaction between the pro-apoptotic factor phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) and autophagy-related protein 5 (ATG5), as well as their regulatory mechanisms in TNBC cell apoptosis and autophagy, to identify potential therapeutic targets for TNBC.
TNBC-related datasets were retrieved from The Cancer Genome Atlas and selected by Prediction Analysis of Microarray 50 analysis to assess the expression of PMAIP1 in the samples. Additionally, the expression of PMAIP1 in the TNBC cell lines (MDA-MB-231) was detected using quantitative real-time polymerase chain reaction. In MDA-MB-231 cells, the expression of PMAIP1 and ATG5 was overexpressed or knocked down, and autophagy was inhibited using chloroquine (20 μM). Gene and protein expression levels were evaluated using quantitative real-time polymerase chain reaction and Western blot, respectively. Immunofluorescence was used to observe microtubule-associated protein 1 light chain 3 puncta formation to assess autophagy levels. Furthermore, cell apoptosis, proliferation, migration, and invasion were analyzed using terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, colony formation assay, and Transwell assay.
Compared to the control group, the expression of PMAIP1 was significantly elevated in TNBC tissues and MDA-MB-231 cells. Furthermore, overexpression of PMAIP1 led to a marked increase in apoptosis levels and a remarkable reduction in autophagy levels in MDA-MB-231 cells, while knockdown of PMAIP1 showed the opposite effects. Additionally, knockdown of ATG5 expression or treatment with chloroquine not only resulted in an increase in PMAIP1 expression in a time-dependent manner, but also reduced autophagy levels and enhanced apoptosis levels of cells. Furthermore, simultaneous knockdown of PMAIP1 and ATG5 considerably up-regulated apoptosis levels while down-regulating autophagy levels. Moreover, knockdown of PMAIP1 alone promoted the viability, invasion, and migration abilities of TNBC cells, while dual knockdown reversed these effects.
Inhibition of ATG5-mediated autophagy maintains PMAIP1 stability, thereby promoting cell apoptosis and suppressing TNBC progression.
三阴性乳腺癌（TNBC）是一种侵袭性很强的乳腺癌亚型，其特点是复发率高且缺乏有效的靶向治疗方法。本研究旨在探讨促凋亡因子佛波醇-12-肉豆蔻酸酯-13-乙酸酯诱导蛋白1（PMAIP1）与自噬相关蛋白5（ATG5）之间的相互作用，以及它们在TNBC细胞凋亡和自噬中的调控机制，以确定TNBC潜在的治疗靶点。
从癌症基因组图谱中检索TNBC相关数据集，并通过微阵列50分析的预测分析进行筛选，以评估样本中PMAIP1的表达。此外，使用定量实时聚合酶链反应检测PMAIP1在TNBC细胞系（MDA-MB-231）中的表达。在MDA-MB-231细胞中，过表达或敲低PMAIP1和ATG5的表达，并使用氯喹（20 μM）抑制自噬。分别使用定量实时聚合酶链反应和蛋白质免疫印迹法评估基因和蛋白质表达水平。使用免疫荧光观察微管相关蛋白1轻链3斑点的形成，以评估自噬水平。此外，使用末端脱氧核苷酸转移酶dUTP缺口末端标记法、集落形成试验和Transwell试验分析细胞凋亡、增殖、迁移和侵袭情况。
与对照组相比，PMAIP1在TNBC组织和MDA-MB-231细胞中的表达显著升高。此外，PMAIP1的过表达导致MDA-MB-231细胞凋亡水平显著增加，自噬水平显著降低，而敲低PMAIP1则显示出相反的效果。此外，敲低ATG5表达或用氯喹处理不仅导致PMAIP1表达随时间依赖性增加，还降低了细胞的自噬水平并增强了细胞的凋亡水平。此外，同时敲低PMAIP1和ATG5可显著上调凋亡水平，同时下调自噬水平。此外，单独敲低PMAIP1可促进TNBC细胞的活力、侵袭和迁移能力，而双重敲低则可逆转这些作用。
抑制ATG5介导的自噬可维持PMAIP1的稳定性，从而促进细胞凋亡并抑制TNBC进展。
