Here, western blot analysis showed that the expression of p-GSK3B and p-SMAD3 in 22RV1 cells (Figure 4d,e) and PC-3 tumor cells (Figure 4f,g) was significantly upregulated compared to that in RWPE-2 cells, suggesting that the EPHB1-GSK3B-SMAD3 signaling pathway was activated during PRAD development. This evidence concerns the gene SMAD3 and prostate adenocarcinoma.