QS290. Targeted Inhibition of the Phosphatidylinositol 3-Kinase Pathway Decreases Colon Cancer Migration

Abstract

Introduction: A better understanding of the signaling pathways contributing to invasion and metastasis of colorectal cancer (CRC) is a prerequisite to designing novel prevention and treatment strategies. The phosphatidylinositol 3-kinase (PI3K) pathway, including its downstream effector proteins, Akt, mTOR, and p70S6K, is recognized as a key modulator of cellular migration and invasion; however, the effects of specific PI3K subunits and Akt isoforms in CRC have not been studied. Methods: Human CRC cells HCT116 and HT29 were transfected with short interfering RNAs (siRNAs) directed at p110α (PI3K catalytic subunit), p85α (PI3K regulatory subunit), Akt1, Akt2, and a non-targeting control sequence (NTC) or treated with selective PI3K (LY294002; 20 μM) or mTOR (rapamycin; 10-50 nM) inhibitors. Migration was evaluated in collagen-coated chemotaxis chamber assays using epidermal growth factor (EGF) or conditioned media from colonic myofibroblasts as the chemoattractant. Cells that migrated were stained and counted under a microscope, and the average number of cells per high power field from triplicate wells was compared to control conditions (assigned 100% or 1.0). Western blotting of cell lysates was performed for p110α, p85α, Akt1, Akt2, and for phosphorylated forms of Akt, mTOR, and p70S6K. Results: Stimulated migration of HCT116 and HT29 cells was decreased by 60-70% after RNA interference of all components including p110α, p85α, Akt1 and Akt2 compared to the NTC sequence (see Fig. A, HT29 cell migration after siRNA treatment). The inhibitors LY294002 and rapamycin decreased migration by 60% and 58%, respectively. Effective knockdown of p110α, p85α, Akt1 and Akt2 with siRNA (Fig. B) and decreased activation of Akt, mTOR, and p70S6K after chemical inhibition were confirmed by Western blotting. Conclusions: Targeted inhibition of the PI3K catalytic and regulatory subunits and both Akt isoforms significantly decreases colon cancer cell migration. We demonstrate that multiple levels of the PI3K/Akt pathway are integral to growth-factor stimulated CRC migration, and that targeting any of these components individually may be an effective strategy for the treatment of metastatic CRC.