Figure 9. Phosphorylation of GIV at S245 is essential for the tumor-suppressive actions of AICAR and Metformin.
(A, B) Parental DLD1 colorectal cancer cells and those stably expressing GIV-WT and the non-phosphorylatable SA and LP mutant GIV constructs were analyzed for their ability to form colonies in soft agar for 2–3 weeks in the presence of increasing concentrations of AICAR (A) or Metformin (B). The number of colonies was counted by light microscopy throughout the depth of the matrix in 15 randomly chosen fields. Graphs display the % reduction in colonies (Y axis) in each cell line, normalized to their respective controls (no treatment). Error bars represent mean ± S.E.M; n = 3; **p<0.01; ***p<0.001; ****p<0.0001. The growth suppressive effects of AICAR (A) and Metformin (B) observed in DLD1 cells expressing GIV-WT were significantly reduced in cells expressing GIV-SA or GIV-LP. (C,D) Working models for how the AMPK-GIV axis impacts cell polarity in normal epithelial cells and in cancers. In the presence of a functional AMPK-GIV axis (C) in which GIV can be phosphorylated by AMPK at S245, cell polarity and the integrity of TJs are preserved in the face of energetic stress, thereby maintaining epithelial barrier function and homeostasis and anchorage-dependency for growth. In the absence of a functional AMPK-GIV axis (D), either because AMPK cannot be activated or GIV cannot be phosphorylated by AMPK at S245, TJs succumb to energetic stress, cell polarity is lost, and cells can undergo anchorage-independent sustained growth.