Pyk2 silencing reduces MM cell proliferation, adhesion, and cell-cycle progression by suppressing Wnt/β-catenin signaling. (A) GFP⁺/Luc⁺-MM.1S Pyk2-knockdown cells were cultured alone or in the presence of MM-BMSCs for 48 hours. Cell proliferation was measured using [³H]-thymidine uptake assay. Inhibition of Pyk2 significantly reduced MM cell proliferation in the presence or absence of MM-BMSCs (P < .0001). (B) The cell cycle of Pyk2-knockdown GFP⁺/Luc⁺-MM.1S cells was assessed by propidium iodine staining and flow-cytometric analysis. Inhibition of Pyk2 increased the G1 phase population from 35.82% in scramble control cells to 42.5% in cells expressing shRNA A2 and 59.8% in those expressing shRNA A4. (C-D) GFP⁺/Luc⁺-MM.1S Pyk2-knockdown cells were cultured in MM-BMSC–coated or fibronectin-coated wells for 2 hours. Inhibition of Pyk2 impaired the adhesion ability of MM cells to BMSCs (P = .0004) and fibronectin (P = .0016). (E) GFP⁺/Luc⁺-MM.1S Pyk2-knockdown cells were cultured alone or in the presence of MM-BMSCs. After coculture of MM cells with primary BMSCs, MM cells were separated by BMSCs, lysed, and subjected to western blot analysis. Inhibition of Pyk2 reduced β-catenin expression through inducing phosphorylated degradation of β-catenin by decreasing p-GSK3β, thus leading to downregulation of c-Myc and Cyclin D1. (F) Decreased mRNA levels of Pyk2, β-catenin, and c-Myc in BM specimens harvested from mice that were injected with Pyk2-knockdown cells. (G) MM-BMSCs enhanced Pyk2 phosphorylation and β-catenin nuclear translocation and upregulated c-Myc at the nuclear level. These effects triggered by BMSCs were abolished by inhibition of Pyk2. (H) Phosphorylation of Src and Paxillin was inhibited with the inhibition of Pyk2. K.D., knockdown; scr, scramble control.