Wee1 inhibitor PD0166285 sensitized TP53 mutant lung squamous cell carcinoma to cisplatin via STAT1

IF 5.3 2区 医学 Q1 ONCOLOGY Cancer Cell International Pub Date : 2024-09-13 DOI:10.1186/s12935-024-03489-w
Qi Li, Wenjie Yang, Qingyi Zhang, Daoming Zhang, Jun Deng, Binxin Chen, Ping Li, Huanqi Zhang, Yiming Jiang, Yangling Li, Bo Zhang, Nengming Lin
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Abstract

Lung squamous cell carcinoma (LUSCs) is associated with high mortality (20–30%) and lacks of effective treatments. Almost all LUSC exhibit somatic mutations in TP53. Wee1, a tyrosine kinase, regulates the cell cycle at the G2/M checkpoint. In TP53-deficient cells, the dependence on G2/M checkpoints increases. PD0166285 is the first reported drug with inhibitory activity against both Wee1 and PKMYT1. Protein expression was determined by Western blot analysis. Cell proliferation was assessed using cell colony formation and CCK-8 assays. Cell cycle was performed by PI staining with flow cytometry. Apoptosis was evaluated using Annexin V-Phycoerythrin double staining and flow cytometry. DNA damage was detected through comet assay and immunofluorescence assay. In vivo, apoptosis and anti-tumor effects were assessed using the TUNEL assay, a nude mouse model, and immunohistochemistry (IHC). Co-immunoprecipitation assay was used to detect protein–protein interactions. We analyzed Wee1, PKMYT1, and Stat1 expression in pan-cancer studies using the Ualcan public database and assessed their prognostic implications with Kaplan–Meier curves. PD0166285, a Wee1 inhibitor, effectively inhibits Wee1 activity, promoting cell entry into a mitotic crisis. Moreover, PD0166285 sensitizes cells to cisplatin, enhancing clinical outcomes. Our study demonstrated that PD016628 regulates the cell cycle through Rad51 and results in cell cycle arrest at the G2/M phase. We observed increased apoptosis in tumor cells treated with PD0166285, particularly when combined with cisplatin, indicating an enhanced apoptotic response. The upregulation of γ-H2AX serves as an indicator of mitotic catastrophe. Co-immunoprecipitation and data analysis revealed that apoptosis in LUSC is mediated through the Stat1 pathway, accompanied by decreased levels of Socs3. Furthermore, IHC staining confirmed significant differences in the expression of Phospho-CDK1 and γ-H2AX in LUSCs, suggesting involvement in DNA damage. In summary, our study suggests that PD0166285, an inhibitor of Wee1, sensitizes LUSC cells to cisplatin and modulates DNA damage and apoptosis pathways through Rad51 and Stat1, respectively. These findings highlight the combination of PD0166285 and cisplatin as a promising therapeutic approach for treating LUSC.
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Wee1 抑制剂 PD0166285 通过 STAT1 使 TP53 突变的肺鳞癌对顺铂敏感
肺鳞状细胞癌(LUSCs)死亡率高(20%-30%),且缺乏有效的治疗方法。几乎所有肺鳞状细胞癌都表现出 TP53 的体细胞突变。Wee1是一种酪氨酸激酶,在G2/M检查点调节细胞周期。在 TP53 缺失的细胞中,对 G2/M 检查点的依赖性增加。PD0166285 是首个报道的同时对 Wee1 和 PKMYT1 具有抑制活性的药物。蛋白表达通过 Western 印迹分析确定。细胞增殖采用细胞集落形成和 CCK-8 检测法进行评估。细胞周期通过流式细胞仪进行 PI 染色。细胞凋亡通过 Annexin V-Phycoerythrin 双染色和流式细胞仪进行评估。DNA 损伤通过彗星试验和免疫荧光试验进行检测。体内凋亡和抗肿瘤效果通过 TUNEL 试验、裸鼠模型和免疫组织化学(IHC)进行评估。共免疫共沉淀试验用于检测蛋白质与蛋白质之间的相互作用。我们利用 Ualcan 公共数据库分析了泛癌症研究中 Wee1、PKMYT1 和 Stat1 的表达情况,并利用 Kaplan-Meier 曲线评估了它们对预后的影响。Wee1抑制剂PD0166285能有效抑制Wee1的活性,促进细胞进入有丝分裂危机。此外,PD0166285还能使细胞对顺铂敏感,从而提高临床疗效。我们的研究表明,PD016628 通过 Rad51 调节细胞周期,导致细胞周期停滞在 G2/M 期。我们观察到用 PD0166285 治疗的肿瘤细胞凋亡增加,尤其是与顺铂联合治疗时,表明凋亡反应增强。γ-H2AX 的上调可作为有丝分裂灾难的指标。共免疫沉淀和数据分析显示,LUSC 的细胞凋亡是通过 Stat1 途径介导的,同时伴随着 Socs3 水平的降低。此外,IHC 染色证实,LUSCs 中 Phospho-CDK1 和 γ-H2AX 的表达存在显著差异,表明其参与了 DNA 损伤。总之,我们的研究表明,Wee1抑制剂PD0166285可使LUSC细胞对顺铂敏感,并分别通过Rad51和Stat1调节DNA损伤和凋亡通路。这些研究结果突出表明,PD0166285与顺铂的结合是治疗LUSC的一种很有前景的治疗方法。
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来源期刊
CiteScore
10.90
自引率
1.70%
发文量
360
审稿时长
1 months
期刊介绍: Cancer Cell International publishes articles on all aspects of cancer cell biology, originating largely from, but not limited to, work using cell culture techniques. The journal focuses on novel cancer studies reporting data from biological experiments performed on cells grown in vitro, in two- or three-dimensional systems, and/or in vivo (animal experiments). These types of experiments have provided crucial data in many fields, from cell proliferation and transformation, to epithelial-mesenchymal interaction, to apoptosis, and host immune response to tumors. Cancer Cell International also considers articles that focus on novel technologies or novel pathways in molecular analysis and on epidemiological studies that may affect patient care, as well as articles reporting translational cancer research studies where in vitro discoveries are bridged to the clinic. As such, the journal is interested in laboratory and animal studies reporting on novel biomarkers of tumor progression and response to therapy and on their applicability to human cancers.
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