Introduction: Pancreatic ductal adenocarcinoma is one of the most brutal types of cancer, and because of its early development of resistance to standard therapeutic agents, and its late diagnosis, its imperative to identify and validate new biomarkers and therapeutic targets.
A lot of evidence relates disturbances in cAMP cascade and PDAC, making this second messenger signaling pathway potentially oncogenic in this setting. To the already known classical mechanisms of regulation of cAMP, it is recently added its extrusion to the extracellular compartment mediated principally by MRP4. In this way, we described the key role of MRP4 in the regulation of cAMP levels in acute myeloid leukemia cells suggesting that this transporter may represent a new potential target for leukemia differentiation therapy. Since then, numerous works arose describing the antiproliferative effects of MRP4-inhibtion, its potential as a marker of poor prognosis and its up-regulation in several tumors.
Regarding PDAC, we have newly described a deregulation in the expression of MRP4 that correlates directly with cell dedifferentiation. The regulatory effect produced by silencing this transporter alters cAMP efflux, and significantly correlates with proliferative and migratory processes (unpublished data).
The aim of this work was to study in vivo effects of MRP4 inhibition in PDAC human xenografts and to explore the expression of MRP4 in human PDAC samples in order to validate MRP4 as a new therapeutic target and biomarker.
Methods: Human tumor xenografts were established by injecting subcutaneously immunodeficient mice with PANC-1 cells expressing two different selected clones for shRNA against MRP4; scrambled was used as control. Tumor size was measured three times per week during 35 days.
Human PDAC samples, including primary and metastatic setting, were included in the analysis. IHC for MRP4, Pan-CK, apoptotic and proliferation markers, were performed.
Results: Silencing MRP4 strongly reduced tumor growth in both animal models. Moreover, the incidence of palpable tumors was greatly decreased in silenced xenografts, indicating a loss of tumorigenicity of these cells. When comparing both silenced clones, effects on both tumor growth and incidence strictly correlated with the expression level of MRP4. Routine H&E evaluation revealed significant differences regarding the amount of epithelial neoplastic cells, apoptotic bodies and mitosis between silenced and control tumors. These findings were confirmed by IHC for Pan-CK, tunnel and ki67 respectively.
Although the apoptotic index was similar in the three clones, the cell exchange ratio (n° mitosis/n° apoptosis) was significantly lower in the MRP4-silenced clones indicating that cells with lower MRP4 expression, proliferate less and die more. Regarding human PDAC samples, MRP4 was expressed in 100% (8/8) of the individuals with disregard of the clinical stage of the disease. We found a clear association between the staining intensity of membranous MRP4 and ki67, suggesting that there is a subset of cells within the tumor that display more MRP4 and which are actively proliferating.
Conclusion: Our results allows for validating MRP4 as a new therapeutic target, and as a possible biomarker for PDAC. Retrospective studies with human samples are being carried out with a bigger number of patients.