背景:研究证实,上皮-间质转化(EMT)和癌症干细胞(CSC)样特性有利于癌症转移。近年来,睾丸特异性蛋白酶50(TSP50)已被确定为预后因子,并参与肿瘤发生调节。然而,TSP50在EMT和CSC样特性维持中的作用和分子机制尚不清楚.
方法:从公共数据库中挖掘TSP50在乳腺癌中的表达和预后价值,并使用生物信息学分析进行探索。然后通过定量RT-PCR(qRT-PCR)进一步验证TSP50及相关基因的表达,蛋白质印迹,免疫组织化学(IHC)。为了探讨TSP50在乳腺癌中的作用,进行了函数损耗和增益实验,在体外和体内。此外,进行免疫荧光(IF)和免疫沉淀(IP)测定以探索TSP50的潜在分子机制。最后,应用IHC技术分析TSP50及相关基因在乳腺癌组织芯片中的表达与临床病理特征的相关性。
结果:TSP50与乳腺癌患者的预后呈负相关。TSP50促进乳腺癌细胞和小鼠异种移植肿瘤组织中的CSC样性状和EMT。此外,抑制PI3K/AKT部分逆转了TSP50诱导的CSC样特性的激活,EMT和肿瘤发生。机械上,TSP50和PI3Kp85α调节亚基可与PI3Kp110α催化亚基竞争性相互作用,促进p110α酶活性,从而激活PI3K/AKT信号通路以维持CSC样表型和促进EMT。此外,人乳腺癌标本的IHC分析显示,TSP50表达与p-AKT和ALDH1蛋白水平呈正相关。值得注意的是,乳腺癌临床病理特征,比如病人的生存时间,肿瘤大小,Ki67,病理分期,N级,雌激素受体(ER)和孕激素受体(PR)水平,与TSP50/p-AKT/ALDH1表达状态相关。
结论:验证了TSP50对EMT和CSC样性质促进的作用依赖于PI3Kp110α。一起,我们的研究揭示了TSP50促进乳腺癌进展的新机制,这可以为基于TSP50的乳腺癌治疗策略提供新的见解。
BACKGROUND: Studies have confirmed that epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC)-like properties are conducive to cancer metastasis. In recent years, testes-specific protease 50 (TSP50) has been identified as a prognostic factor and is involved in tumorigenesis regulation. However, the role and molecular mechanisms of TSP50 in EMT and CSC-like properties maintenance remain unclear.
METHODS: The expression and prognostic value of TSP50 in breast cancer were excavated from public databases and explored using bioinformatics analysis. Then the expression of TSP50 and related genes was further validated by quantitative RT-PCR (qRT-PCR), Western blot, and immunohistochemistry (IHC). In order to investigate the function of TSP50 in breast cancer, loss- and gain-of-function experiments were conducted, both in vitro and in vivo. Furthermore, immunofluorescence (IF) and immunoprecipitation (IP) assays were performed to explore the potential molecular mechanisms of TSP50. Finally, the correlation between the expression of TSP50 and related genes in breast cancer tissue microarray and clinicopathological characteristics was analyzed by IHC.
RESULTS: TSP50 was negatively correlated with the prognosis of patients with breast cancer. TSP50 promoted CSC-like traits and EMT in both breast cancer cells and mouse xenograft tumor tissues. Additionally, inhibition of PI3K/AKT partly reversed TSP50-induced activation of CSC-like properties, EMT and tumorigenesis. Mechanistically, TSP50 and PI3K p85α regulatory subunit could competitively interact with the PI3K p110α catalytic subunit to promote p110α enzymatic activity, thereby activating the PI3K/AKT signaling pathway for CSC-like phenotypes maintenance and EMT promotion. Moreover, IHC analysis of human breast cancer specimens revealed that TSP50 expression was positively correlated with p-AKT and ALDH1 protein levels. Notably, breast cancer clinicopathological characteristics, such as patient survival time, tumor size, Ki67, pathologic stage, N stage, estrogen receptor (ER) and progesterone receptor (PR) levels, correlated well with TSP50/p-AKT/ALDH1 expression status.
CONCLUSIONS: The effects of TSP50 on EMT and CSC-like properties promotion were verified to be dependent on PI3K p110α. Together, our study revealed a novel mechanism by which TSP50 facilitates the progression of breast cancer, which can provide new insights into TSP50-based breast cancer treatment strategies.