Supplementary Material for: PTEN Loss-Mediated Akt Activation Increases the Properties of Cancer Stem-Like Cell Populations in Prostate Cancer

Objective: To demonstrate that the PTEN/PI3K/Akt/NF-κB pathway plays an important role in regulating the prostate cancer stem-like cell population by upregulating ABCG2. Methods: Targeted PTEN knockdown in human prostate DU145 and 22Rv1 cells using a small interfering RNA were confirmed by immunoblot analysis using antibodies of PTEN, phospho-Akt, Akt, and a-tubulin. Knockdown PTEN DU145 and 22Rv1 cells were augmented, and the stem cell-like properties were examined by cell viability and tumor sphere formation and treated by Akt IV inhibitor to provide the signal transduction pathway. Luciferase activity assays were performed. Results: The knockdown of PTEN in prostate cancer cell lines increased the stem-like properties of the cells, including their sphere-forming ability, stem cell population number, epithelial-mesenchymal transition-related gene expression, and ABCG2 expression. Additionally, PTEN expression was highly associated with elevated expression of phospho-Akt. Treatment with an Akt inhibitor suppressed the PTEN-mediated effects on the properties of these stem-like cells as well as drug resistance, ABCG2 expression, and the NF-κB pathway. Conclusion: The loss of PTEN in prostate cancer cells resulted in an increased PI3K/Akt pathway. Due to the Akt activation, PTEN loss may play an important role in prostate cancer by promoting cancer stemness through a mechanism that involves enhanced NF-κB signaling.

研究目的：验证PTEN/PI3K/Akt/NF-κB信号通路通过上调ABCG2，在调控前列腺癌干细胞样细胞群体中发挥重要作用。研究方法：采用小分子干扰RNA（small interfering RNA）对人前列腺DU145与22Rv1细胞进行靶向PTEN敲低，通过免疫印迹分析（immunoblot analysis），使用PTEN、磷酸化Akt（phospho-Akt）、Akt以及α-微管蛋白（α-tubulin）对应的抗体验证敲低效果。对敲低PTEN的DU145与22Rv1细胞进行扩增培养，通过细胞活力检测、肿瘤球形成实验评估其干细胞样特性，并使用Akt IV抑制剂处理以解析该信号转导通路；同时开展荧光素酶活性检测实验（luciferase activity assays）。研究结果：前列腺癌细胞系中PTEN敲低可增强细胞的干细胞样特性，包括球形成能力、干细胞群体数量、上皮间质转化（epithelial-mesenchymal transition, EMT）相关基因表达以及ABCG2表达水平。此外，PTEN表达与磷酸化Akt的升高呈显著正相关。Akt抑制剂处理可抑制PTEN介导的上述干细胞样细胞特性改变，同时逆转其耐药性、ABCG2表达上调以及NF-κB通路激活。研究结论：前列腺癌细胞中PTEN缺失会导致PI3K/Akt通路激活。由于Akt的活化，PTEN缺失可能通过增强NF-κB信号通路的机制，促进肿瘤干细胞干性，进而在前列腺癌发生发展中发挥重要作用。