目的:食管鳞状细胞癌(ESCC)是一种侵袭性疾病,预后不良,由关键细胞生长调节剂的失活引起,导致不受控制的增殖和恶性肿瘤增加。虽然丝氨酸/苏氨酸激酶3(STK3),也称为哺乳动物STE20样蛋白激酶2(MST2),是Hippo途径的一种高度保守的激酶,在免疫调节中起着至关重要的作用,器官发育,细胞分化,和癌症抑制,其在ESCC中的表型和功能需要进一步研究。在这项研究中,我们首次报道了STK3激酶在ESCC中的作用及其活化条件,以及激酶激活的机制和介质。
方法:在本研究中,我们研究了STK3在ESCC中的表达和临床意义。我们首先使用生物信息学数据库和免疫组织化学来分析ESCC患者队列中的STK3表达并进行生存分析。在体内,我们使用裸鼠模型进行了致瘤性测定,以证明STK3激酶的表型。体外,我们进行了蛋白质印迹分析,qPCR分析,CO-IP,和免疫荧光(IF)染色分析以检测分子表达,互动,和分配。我们测量了增殖,迁移,使用CCK-8和transwell测定实验组中ESCC细胞的凋亡能力,流式细胞术,和EdU染色。我们使用RNA-seq来鉴定在沉默的STK3或FOXO1的ESCC细胞中差异表达的基因。我们在体外使用Western印迹和ChIP证明了STK3-FOXO1轴介导的TP53INP1/P21基因的调控关系。
结果:我们证明了与食管上皮相比,ESCC组织和细胞系中STK3的高表达。细胞ROS诱导ESCC细胞中的STK3自磷酸化,导致p-STK3/4上调。STK3活化通过触发凋亡和抑制细胞周期来抑制ESCC细胞增殖和迁移。STK3激酶激活磷酸化FOXO1Ser212,促进核易位,增强转录活性,并上调TP53INP1和P21。我们还研究了TP53INP1和P21在ESCC中的表型效应,发现它们的击倒显著增加了肿瘤的增殖,强调它们在ESCC肿瘤发生中的关键作用。
结论:STK3激酶在食管鳞癌中高表达,可被细胞ROS激活,抑制细胞增殖和迁移。此外,STK3激活介导的FOXO1通过靶向TP53INP1/P21调节ESCC细胞凋亡和细胞周期阻滞。我们的研究强调了STK3在ESCC中的抗肿瘤功能,并阐明了其对ESCC的抗肿瘤作用的机制。
OBJECTIVE: Esophageal squamous cell carcinoma (ESCC) is an aggressive disease with a poor prognosis, caused by the inactivation of critical cell growth regulators that lead to uncontrolled proliferation and increased malignancy. Although Serine/Threonine Kinase 3 (STK3), also known as Mammalian STE20-like protein kinase 2 (MST2), is a highly conserved kinase of the Hippo pathway, plays a critical role in immunomodulation, organ development, cellular differentiation, and cancer suppression, its phenotype and function in ESCC require further investigation. In this study, we report for the first time on the role of STK3 kinase and its activation condition in ESCC, as well as the mechanism and mediators of kinase activation.
METHODS: In this study, we investigated the expression and clinical significance of STK3 in ESCC. We first used bioinformatics databases and immunohistochemistry to analyze STK3 expression in the ESCC patient cohort and conducted survival analysis. In vivo, we conducted a tumorigenicity assay using nude mouse models to demonstrate the phenotypes of STK3 kinase. In vitro, we conducted Western blot analysis, qPCR analysis, CO-IP, and immunofluorescence (IF) staining analysis to detect molecule expression, interaction, and distribution. We measured proliferation, migration, and apoptosis abilities in ESCC cells in the experimental groups using CCK-8 and transwell assays, flow cytometry, and EdU staining. We used RNA-seq to identify genes that were differentially expressed in ESCC cells with silenced STK3 or FOXO1. We demonstrated the regulatory relationship of the TP53INP1/P21 gene medicated by the STK3-FOXO1 axis using Western blotting and ChIP in vitro.
RESULTS: We demonstrate high STK3 expression in ESCC tissue and cell lines compared to esophageal epithelium. Cellular ROS induces STK3 autophosphorylation in ESCC cells, resulting in upregulated p-STK3/4. STK3 activation inhibits ESCC cell proliferation and migration by triggering apoptosis and suppressing the cell cycle. STK3 kinase activation phosphorylates FOXO1Ser212, promoting nuclear translocation, enhancing transcriptional activity, and upregulating TP53INP1 and P21. We also investigated TP53INP1 and P21\'s phenotypic effects in ESCC, finding that their knockdown significantly increases tumor proliferation, highlighting their crucial role in ESCC tumorigenesis.
CONCLUSIONS: STK3 kinase has a high expression level in ESCC and can be activated by cellular ROS, inhibiting cell proliferation and migration. Additionally, STK3 activation-mediated FOXO1 regulates ESCC cell apoptosis and cell cycle arrest by targeting TP53INP1/P21. Our research underscores the anti-tumor function of STK3 in ESCC and elucidates the mechanism underlying its anti-tumor effect on ESCC.