Activation of wnt/β-catenin signaling enhanced stemness of prostate cancer cells / 肿瘤

ABSTRACT

Objective: To explore the relationship between the activity of Wnt/β-catenin signaling pathway and prostate cancer stem cells by utilizing a Wnt/β-catenin signaling reporter system. Methods: Immunofluorescence staining was used to explore the activation of Wnt/β-catenin signaling pathway in prostate cancer. Wnt/β-catenin signaling reporter plasmid 7TGP carrying T cell specific transcription factor (TCF) binding site and enhanced green fluorescent protein (GFP) was transfected into prostate cancer PC3 and DU145 cells by liposomes, respectively. Furthermore, the top 5% (GFP+) and bottom 5% (GFP-) groups of PC3 and DU145 cells according to the fluorescent intensity of GFP were collected via flow cytometry. Then Western blotting assay was used to compare the expression level of activated β-catenin protein in the nucleus of GFP+ and GFP- cell groups. The real-time fluorescent quantitative PCR was used to detect the expressions of Wnt/β-catenin signaling downstream target genes in GFP+ and GFPcell groups. Sphere formation assay and real-time fluorescent quantitative PCR were used to compare the stemness of GFP+ and GFP- cells. Results: The activity of canonical Wnt signaling pathway was low in most of prostate cancer cells, in which β-catenin was not activated and translocated to the nucleus. While β-catenin was increasingly translocated to the nucleus in a small percentage of prostate cancer cells resulting in high activation of Wnt signaling pathway. PC3-GFP+ and DU145-GFP+ cells had more expression of nuclear β-catenin comparing with the corresponding GFP- cells (both P < 0.05), and the expression level of Wnt signaling pathway down-stream target gene Axis inhibition protein 2 (AXIN2) was up-regulated (both P < 0.05). In addition, the expression levels of cancer stem cell markers including B-cell-specific moloney leukemia virusinsert site 1 (BMI1) and aldehyde dehydrogenase family 1 member A1 (ALDH1A1) were up-regulated in PC3-GFP+ and DU145-GFP+ cells comparing with the corresponding GFP- cells (all P < 0.05). The sphere formation capacity was remarkedly up-regulated in PC3-GFP+ and DU145-GFP+ cells comparing with the corresponding GFP- cells (both P < 0.05). Conclusion: Prostate cancer cells with higher activity of Wnt/β-catenin signaling show the enhanced stemness.