A novel EPAC-specific inhibitor suppresses pancreatic cancer cell migration and invasion

Abstract

Exchange protein directly activated by cAMP (EPAC) and cAMP-dependent protein kinase (PKA) are two intracellular receptors that mediate the effects of the prototypic second messenger cAMP. Identifying pharmacological probes for selectively modulating EPAC activity represents a significant unmet need within the research field. Herein, we report the identification and characterization of 3-(5-tert-butyl-isoxazol-3-yl)-2-[(3-chloro-phenyl)-hydrazono]-3-oxo-propionitrile (ESI-09), a novel noncyclic nucleotide EPAC antagonist that is capable of specifically blocking intracellular EPAC-mediated Rap1 activation and Akt phosphorylation, as well as EPAC-mediated insulin secretion in pancreatic β cells. Using this novel EPAC-specific inhibitor, we have probed the functional roles of overexpression of EPAC1 in pancreatic cancer cells. Our studies show that EPAC1 plays an important role in pancreatic cancer cell migration and invasion, and thus represents a potential target for developing novel therapeutic strategies for pancreatic cancer.

Fig. 1.

Chemical structure of ESI-09.

Fig. 2.

Relative potency of ESI-09. (A) dose-dependent competition of ESI-09 (open circles) and cAMP (closed squares) with 8-NBD-cAMP in binding to EPAC2. (B) dose-dependent inhibition of EPAC1 (closed circles) or EPAC2 (open circles) GEF activity by ESI-09 in the presence of 25 µM cAMP.

Fig. 3.

Predicted binding mode and molecular docking of ESI-09 into the CBD of EPAC1 protein (homology modeling) and EPAC2 protein (Protein Data Bank code 3CF6). (A) binding mode of ESI-09 with EPAC1 protein (homology modeling); (B) surface of the CBD of EPAC1 protein; (C) binding mode of ESI-09 with EPAC2 protein; (D) surface of the CBD of EPAC2 protein. Important residues are drawn in sticks. Hydrogen bonds are shown as dashed green lines. ESI-09 is shown in pink, and cAMP is shown in pale yellow.

Fig. 4.

ESI-09 inhibits EPAC- but not EGF-mediated Akt phosphorylation in AsPC-1 pancreatic cancer cells. Serum-starved AsPC-1 cells were stimulated with vehicle, 10 µM 007-AM, or 0.1 µM EGF after pretreatment with the indicated concentrations of ESI-09. Cell lysates were subjected to Western blot analyses using anti-phospho-Akt T308 and S473 antibodies (representative blot shown). Data points represent mean ± S.D. (n = 3). #Significantly higher than vehicle group (P < 0.02). *Significantly lower that 007-AM-stimulated group (P < 0.02).

Fig. 5.

ESI-09 inhibits EPAC2-mediated insulin secretion in INS-1 cells. INS-1 cells were stimulated with vehicle or 10 µM 007-AM after pretreatment with the indicated concentrations of ESI-09. Bars represent mean ± S.D. (n = 3). #Significantly higher than vehicle group (P < 0.05). *Significantly lower that 007-AM-stimulated group (P < 0.05).

Fig. 6.

EPAC1 inhibition decreases pancreatic cancer cell migration and invasion. AsPC-1 and PANC-1 cells were pretreated with the indicated concentrations of ESI-09 for 24 hours before migration and invasion were measured by Transwell (A) and wound healing assays (B). EPAC1 expression was suppressed by shEPAC1-C28 and shEPAC1-C32 and migration and invasion were measured by Transwell assays (C). Bars represent mean ± S.D. (n = 3). *Significantly lower than vehicle group (P < 0.05). #Significantly lower than parental cells group (P < 0.02).

Fig. 7.

ESI-09 inhibits EPAC1-mediated adhesion of PDA cells on collagen I. AsPC-1 and PANC-1 cells were stimulated with vehicle or 10 µM 007-AM after treatment with the indicated concentrations of ESI-09 for 5 minutes. Bars represent mean ± S.D. (n = 3). #Significantly higher than vehicle group (P < 0.03). *Significantly lower than 007-AM-stimulated group (P < 0.02).