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

<b><i>Objective:</i></b> 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. <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> 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. <b><i>Conclusion:</i></b> 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.