Signaling by small
guanosine triphosphatases (
GTPase), Rap1/Rac1, is one of the major pathways controlling
cancer cell migration and
tumor metastasis.
Thymosin beta-4 (Tβ4), an actin-sequestering
protein, has been shown to increase migration of
cancer cells. Episodes of
hypoxia and re-oxygenation (H/R) are an important phenomenon in tumor microenvironment (TME). We investigated whether Tβ4 could play as an intermediary to crosstalk between Rac1- and Rap1-
GTPase activation under
hypoxia/reoxygenation (H/R) conditions. Inhibition of Tβ4 expression using
transcription activator-like effector nucleases (
TALEN) significantly decreased lung
metastasis of B16F10 cells. Rac1 and Rap1 activity, as well as
cancer cell migration, increased following induction of Tβ4 expression in normoxia- or H/R-experienced cells, but were barely detectable in Tβ4-depleted cells. Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (
CPT), a Rap1 activator. In contrast, a Rac1-specific inhibitor,
NSC23766, and dominant negative Rac1 (Rac1N17) enhanced Tβ4 expression and aberrant Rap1 activity. While
NSC23766 and Rac1N17 incompletely inhibited
tumor metastasis in vivo, and H/R-experienced
cancer cell migration in vitro, more efficient attenuation of
cancer cell migration was accomplished by simultaneous inactivation of Rap1 and Rac1 with Rap1N17 and Rac1N17, respectively. These data suggest that a combination
therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting
tumor metastasis.