Pharmacological inhibition and genetic knockdown of exchange protein directly activated by cAMP 1 reduce pancreatic cancer metastasis in vivo

Abstract

cAMP plays a critical role in regulating migration of various cancers. This role is context dependent and is determined by which of the two main cAMP sensors is at play: cAMP-dependent protein kinase or exchange protein directly activated by cAMP (EPAC). Recently, we have shown that the cAMP sensor protein EPAC1 promotes invasion/migration of pancreatic ductal adenocarcinoma (PDA) in vitro. In this study, we investigated the role of EPAC1 in invasion and metastasis of PDA in vivo, and evaluated the therapeutic potential of EPAC inhibitors as antimetastasis agents for this neoplasm. We employed an orthotopic metastatic mouse model in which the PDA cells MIA PaCa-2 were injected into the pancreas of athymic nude mice, and their local and distant spread was monitored by in vivo imaging and histologic evaluation of the number of metastatic foci in the liver. Either genetic suppression of EPAC1 or its pharmacologic inhibition with 3-(5-tert-butyl-isoxazol-3-yl)-2-[(3-chloro-phenyl)-hydrazono]-3-oxo-propionitrile, an EPAC-specific antagonist recently identified in our laboratory, decreased invasion and metastasis of the PDA cells. Mechanistically, EPAC1 promotes activation and trafficking of integrin β1, which plays an essential role in PDA migration and metastasis. Our data show that EPAC1 facilitates metastasis of PDA cells and EPAC1 might be a potential novel therapeutic target for developing antimetastasis agents for PDA.

Fig. 1.

EPAC1 inhibition or knockdown decreases invasion and migration of MIA PaCa-2. (A) Cells were treated with the EPAC agonist 007-AM in the presence or absence of the EPAC inhibitor ESI-09, and Rap1 activation (GTP-bound) was probed by Western blotting. (B) An invasion/migration assay showing an increase in invasion/migration of MIA PaCa-2 cells with 007-AM treatment and a decrease by Epac1-KD or ESI-09 treatment. (C) A wound-healing assay showing an increase in wound-closure rate of MIA PaCa-2 cells with 007-AM treatment and a decrease by Epac1-KD or ESI-09 treatment. Wound closure is presented as the distance traveled by the edge of the wound relative to the wound’s initial size. *Significantly higher or lower than vehicle-treated Ctrl MIA PaCa-2 cells (P < 0.03). Bars represent mean ± S.D. (n = 3).

Fig. 2.

Suppression of EPAC1 reduces metastasis of MIA PaCa-2. (A) Cells were injected into the pancreas of athymic nude mice, and invasion/metastasis was monitored in vivo by bioluminescence imaging. The image shown was obtained 3 weeks post< >injection. Arrowheads show signal from the primary tumor and local invasion. (B) Representative image of H&E staining of the liver showing a metastatic focus (micromets) of MIA PaCa-2 cells (arrowhead); scale bar, 10 µm. (C) Quantification of liver micromets (number of micromets/H&E slide). For each mouse, the number of micromets is the average of two slides taken ∼20 µm apart. *Significantly lower than vehicle-treated group (P < 0.02). Bars represent mean ± S.D.

Fig. 3.

EPAC1 increases trafficking of integrin β1 to the plasma membrane. (A and B) MIA PaCa-2 cells were treated with 007-AM in the presence or absence of ESI-09, and total or plasma membrane proteins were isolated, respectively. Itgβ1 levels were probed by Western blotting, quantified by densitometry, and presented as a percentage of the indicated loading control. (C) Cells were trypsinized, and recovery of surface integrin β1 was probed by FACS. Data are presented as mean fluorescence intensity (MFI) and normalized to vehicle-treated Ctrl MIA PaCa-2 cells. **Significantly higher than vehicle-treated Ctrl cells (P < 0.01). *Significantly lower than vehicle-treated Ctrl cells (P < 0.02). Bars represent mean ± S.D. (n = 3).

Fig. 4.

EPAC1 increases trafficking of integrin β1 to the plasma membrane through PKC. MIA PaCa-2 cells were treated with 007-AM in the presence or absence of BIM I. (A and B) Total or plasma membrane proteins were isolated, respectively. Itgβ1 levels were probed by Western blotting, quantified by densitometry, and presented as a percentage of the indicated loading control. (C) Invasion/Migration was examined by a Transwell invasion/migration assay. (D) Wound-healing rate was examined in a wound-healing assay. Wound closure rate is presented as the distance traveled by the edge of the wound relative to the wound’s initial size. *Significantly higher or lower than vehicle-treated cells (P < 0.05). **Significantly higher than vehicle-treated cells (P < 0.02). ^#Significantly lower than 007-AM–treated cells (P < 0.03). Bars represent mean ± S.D. (n = 3).

Fig. 5.

EPAC1 facilitates activation of integrin β1. Cells were treated with 007-AM in the presence or absence of ESI-09 or NPC 15437, and activation of integrin β1 was probed by FACS using the antibody 12G10, which only binds to the active form of integrin β1. Total integrin β1 was probed with the antibody K-20. (A) A representative histogram showing the binding of 12G10. (B) Quantification of active Itgβ1 relative to total Itgβ1 [mean fluorescence intensity (MFI)_active/MFI_total]. **Significantly higher than vehicle-treated Ctrl cells (P < 0.03). *Significantly lower than vehicle-treated Ctrl cells (P < 0.04). Bars represent mean ± S.D. (n = 3).

Fig. 6.

Pharmacologic inhibition of EPAC1 reduces metastasis of MIA PaCa-2. Luciferase-transduced MIA PaCa-2 cells were injected into the pancreas of athymic nude mice, and animals were treated with ESI-09 (daily injection of 10 mg/kg i.p.) or vehicle. (A) In vivo bioluminescence image taken 3 weeks post injection of cells. Arrowheads show signal from the primary tumor and local invasion. (B) Quantification of liver micromets (number of micromets/H&E slide). For each mouse, the number of micromets is the average of two slides taken ∼20 µm apart. *Significantly lower than vehicle-treated group (P < 0.04). Bars represent mean ± S.D.