ADRENOMEDULLIN IS AN AUTOCRINE REGULATOR OF PANCREATIC CANCER ACTING VIA THE ADMR RECEPTOR

Abstract

Background: Pancreatic cancer is a major oncological challenge, due to its aggressive growth and metastasis. We have shown that adrenomedullin (AM) is highly expressed in pancreatic cancer compared to normal pancreas and chronic pancreatitis (Cancer Res. 63:2649, 2003). In the current study, we have investigated the role of AM expression on pancreatic cancer cell function and the receptor involved in its effects. Methods: RT-PCR and IHC confirmed AM expression in human pancreatic tumor samples. AM levels were increased by transfection with an expression vector in Panc-1 cells, which had a low endogenous level, and were reduced by transfection with an AM shRNA construct in MPanc96 cells, which had a high endogenous level. AM levels were verified by ELISA. Growth and invasion were assessed by MTS and Matrigel invasion assays in several pancreatic cancer cell lines in vitro. In vivo studies were conducted by developing orthotopic tumor in the pancreas of Scid mice and tumor growth and invasion were analyzed by bioluminescence imaging. AM receptors on pancreatic cancer, stellate, and endothelial cells were identified by RT-PCR. NF-κB activity was measured by a luciferase reporter study. ADMR was silenced using siRNA. Results: AM was expressed in 90% (43/48) of pancreatic adenocarcinomas as evaluated by IHC. Exogenous addition of AM increased proliferation, invasion, and NFκB activity in all examined pancreatic cancer cells in vitro. The AM antagonist AMA reduced basal pancreatic cancer cell proliferation and NFκB activity in all examined cell lines. AM expression also increased these parameters in Panc-1 cells. Silencing of AM by expression of stable AM shRNA in MPanc96 cells reduced basal levels of proliferation and invasion. In an in vivo orthotopic model, AM over-expressing Panc-1 cells formed more rapidly growing tumors than wild-type cells, while AM-silenced Mpanc96 tumors had decreased tumor growth and invasion compared to wild-type cells. To identify the receptors responsible for these effects of AM on pancreatic cancer cells we measured the mRNA levels for the AM receptors CRLR and ADMR by RT-PCR. ADMR was the only receptor expressed in pancreatic cancer, while both CRLR and ADMR were present in pancreatic stellate cells and endothelial cells. Expression of ADMR was also verified by western blotting. Silencing of ADMR by siRNA reduced pancreatic cancer cell growth in vitro. Conclusions: AM is highly expressed in pancreatic cancer cells and acts as an autocrine factor to stimulate pancreatic cancer cell growth, invasion and NFκB activity in vitro and tumor growth and invasion in vivo. The autocrine effects of AM on pancreatic cancer appear to be mediated by the ADMR receptor. AM and or the ADMR receptor may be useful targets for the development of novel therapies for pancreatic cancer.