Identification of MRP4/ABCC4 as a Target for Reducing the Proliferation of Pancreatic Ductal Adenocarcinoma Cells by Modulating the cAMP Efflux

Abstract

Pancreatic cancer is one of the most lethal types of tumors with no effective therapy available; is currently the third leading cause of cancer in developed countries; and is predicted to become the second deadliest cancer in the United States by 2030. Due to the marginal benefits of current standard chemotherapy, the identification of new therapeutic targets is greatly required. Considering that cAMP pathway is commonly activated in pancreatic ductal adenocarcinoma (PDAC) and its premalignant lesions, we aim to investigate the multidrug resistance-associated protein 4 (MRP4)-dependent cAMP extrusion process as a cause of increased cell proliferation in human PDAC cell lines. Our results from in silico analysis indicate that MRP4 expression may influence PDAC patient outcome; thus, high MRP4 levels could be indicators of poor survival. In addition, we performed in vitro experiments and identified an association between higher MRP4 expression levels and more undifferentiated and malignant models of PDAC and cAMP extrusion capacity. We studied the antiproliferative effect and the overall cAMP response of three MRP4 inhibitors, probenecid, MK571, and ceefourin-1 in PDAC in vitro models. Moreover, MRP4-specific silencing in PANC-1 cells reduced cell proliferation (P , 0.05), whereas MRP4 overexpression in BxPC-3 cells significantly incremented their growth rate in culture (P , 0.05). MRP4 pharmacological inhibition or silencing abrogated cell proliferation through the activation of the cAMP/Epac/Rap1 signaling pathway. Also, extracellular cAMP reverted the antiproliferative effect of MRP4 blockade. Our data highlight the MRP4-dependent cAMP extrusion process as a key participant in cell proliferation, indicating that MRP4 could be an exploitable therapeutic target for PDAC. SIGNIFICANCE STATEMENTABCC4/MRP4 is the main transporter responsible for cAMP efflux. In this work, we show that MRP4 expression may influence PDAC patient outcome and identify an association between higher MRP4 expression levels and more undifferentiated and malignant in vitro models of PDAC. Findings prove the involvement of MRP4 in PDAC cell proliferation through a novel extracellular cAMP mitogenic pathway and further support MRP4 inhibition as a promising therapeutic strategy for PDAC treatment.

Constitutive activity of dopamine receptor type 1 (D1R) increases CaV2.2 currents in PFC neurons

“…FRET time course of cAMP intracellular levels was measured as previously described ( Carozzo et al, 2019 ). Briefly, HEKT Epac-S H187 cells transfected with D1R, D1R plus D1R loop 2, or empty pcDNA3.1 (+) plasmid was seeded in 96-well plates at a density of 10 5 cells per well.…”

The Regulator of G Protein Signaling Homologous Domain of G Protein-Coupled Receptor Kinase 2 Mediates Short-Term Desensitization of β3-Adrenergic Receptor

“…FRET time-course of cAMP intracellular levels was measured as previously described (Carozzo et al, 2019). Briefly, HEKT Epac-S H187 transfected with pCDNA3.1 HA-b3-adrenergic receptor alone or with pCEFL GRK2 pC were seeded in 96-well plates at a density of 1 x 10 5 cells/well.…”

Roles of ABCC1 and ABCC4 in Proliferation and Migration of Breast Cancer Cell Lines

“…This may be due to their ability to export chemotherapeutic drugs, leading to multidrug resistance [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. However, ABCC1 and ABCC4 can also transport a wide range of substrates involved in inflammation and cellular signaling and have been shown in several different cancer types to have a role in cancer development and progression that is independent of drug resistance [ 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 55 ]. Therefore, in this study, we investigated whether ABCC1 or ABCC4 are involved in cellular proliferation or migration in breast cancer cell lines.…”

“…This contrasts with a study investigating the role of these transporters in neuroblastoma, where ABCC4 was more associated with proliferation and ABCC1 with migration [ 39 ]. Similarly, in pancreatic cancer, ABCC4 was associated with proliferation [ 47 ]. However, in epithelial ovarian cancer, ABCC4 was associated with proliferation, migration, and invasion [ 45 ].…”

“…Studies on neuroblastoma (a rare childhood cancer) have confirmed that both ABCC1 and ABCC4 play an important physiological role in its development, independent of their role in multidrug resistance, affecting cellular proliferation, migration, and differentiation [ 39 ]. ABCC4 has also been implicated in cancer cell proliferation in leukemia [ 40 , 41 ], gastric cancer [ 42 ], lung cancer [ 43 ], renal cancer [ 44 ], ovarian cancer [ 45 ], and pancreatic cancer [ 46 , 47 ]. However, less is known about whether ABCC1 and ABCC4 have a role in breast cancer development and/or progression.…”