Figure 5

AMP-activated protein kinase (AMPK)-phosphoprotein enriched in astrocytes 15 kDa (PEA15) axis is critical for tumorigenicity of breast cancer cells. (A) Breast cancer cell line derived from metastatic site (pleural effusion) (MDAMB231) cells were transfected with control small interfering RNA (siRNA) or siRNA targeting AMPK α2 and injected subcutaneously (1 × 10⁶ cells/injection) into six nude mice (control siRNA transfected cells in the left flank, and AMPK α2 siRNA-transfected cells in the right flank) and monitored for tumor formation for seven weeks. (B and C) MDAMB231 cells (B) and BT 474 cells (C) stably expressing short hairpin RNA (shRNA) pool against AMPK α2 or scrambled shRNA were introduced subcutaneously (1 × 10⁶ cells/injection) into three nude mice and tumor formation was monitored for the indicated time period. Control shRNA-expressing cells were injected in the left flank, and AMPK α2 shRNA-expressing cells in the right flank. Tumors were resected and tumor-derived cells subjected to immunoblotting for specified proteins. (D and E) MDAMB231 cells (D) and BT 474 cells (E) stably expressing CSCG-WT-Flag PEA15 or CSCG-S116A-Flag-PEA15 were injected subcutaneously (1 × 10⁶ cells/injection) into six nude mice and tumor formation was monitored for the indicated time period. Cells stably expressing wild-type (WT)-PEA15 were injected in the left flank, while cells stably expressing S116A-PEA15 were injected in the right flank. Tumors were resected and tumor-derived cells subjected to immunoblotting for specified proteins.