Fig 1: TFAP2A-Dependent Effect of SUMO Inhibition in Colorectal Cancer Cell Lines and Primary Colorectal Tumors(A) HCT116 cells were transfected with siRNA to UBC9 or PIAS1 alone or in combination with siRNA to TFAP2A to demonstrate effect on CD44 RNA (**p < 0.01) (top panel) and CD44, TFAP2A, UBC9, and PIAS1 protein (bottom). Graph of RNA data was from three independent experiments. Relative CD44 protein: NT knockdown (KD), 1.0; TFAP2A KD, 0.98; UBC9 KD, 0.48; PIAS1 KD, 0.35; TFAP2A + UBC9 KD, 1.0; TFAP2A + PIAS1 KD, 0.91. Error bars represent the SE.(B) HCT116 cells were transfected with empty vector (EV), expression vectors for TFAP2A wild-type (WT), or K10R mutant (K10R) and western blot was performed after 96 hr. Relative TFAP2A protein: EV, 1.0; TFAP2A, 1.8; K10R, 2.2. Relative CD44 protein: EV, 1.0; TFAP2A, 1.09; K10R, 0.28. Relative MMP14 protein: EV, 1.0; TFAP2A, 0.98; K10R, 0.49.(C) CD44 RNA from three tumor isolates treated with ginkgolic or anacardic acid in vitro (***p < 0.001). Inset: example of western blot from primary colorectal tumor cells. Relative CD44 protein: vehicle (VEH), 1.0, anacardic acid (AA), 0.02.(D) FACS analysis of CD44 and CD166/ALCAM expression in primary colon cancer cells treated with anacardic acid or vehicle. Percentage of cells expressing CSC phenotypic markers CD44+/hiCD166+/hi was reduced from 11% to 2% with anacardic acid treatment.