Adrenomedullin is induced by hypoxia and enhances pancreatic cancer cell invasion

Abstract

Adrenomedullin (ADM) is synthesized by different types of cells and acts by binding calcitonin receptor-like receptor (CRLR) and members of the receptor activity-modifying protein (RAMP) family. In this study, the expression and functional role of ADM and its signaling components were investigated in pancreatic adenocarcinoma (PDAC). By QRT-PCR, median mRNA levels of ADM and CRLR were 1.5-and 2.4-fold higher, respectively, in PDAC tissues compared to normal pancreatic tissues. By immunohistochemistry, ADM, CRLR, RAMP1 and RAMP2, but not RAMP3, were expressed in pancreatic cancer cells. ADM serum levels were significantly increased in PDAC patients compared to healthy controls and chronic pancreatitis (CP) patients, with an area under the ROC curve of 0.83 and 0.98, respectively. At a cut-off level of 30.6 ng/ml, the specificity of ADM to differentiate PDAC from controls and CP patients was 85.5 and 83.6%, with a sensitivity of 80 and 100%. All 5 evaluated pancreatic cancer cells lines expressed ADM, CRLR, RAMP1 and RAMP2, whereas RAMP3 was expressed in only 1/5 pancreatic cancer cell lines. ADM was strongly induced by hypoxia and significantly increased invasiveness in 3/5 human pancreatic cancer cells. Blocking of CRLR decreased invasiveness in 4/5 human pancreatic cancer cells. In addition, rADM slightly up-regulated vascular endothelial growth factor secretion in 3/5 cell lines. In conclusion, ADM is induced by hypoxia and over-expressed in PDAC and might therefore serve as a potential tumor marker. Furthermore, ADM increases invasiveness of some pancreatic cancer cells and might influence angiogenesis, suggesting that blocking this pathway might have a therapeutic potential. ' 2007 Wiley-Liss, Inc.Key words: pancreatic cancer; hypoxia; invasion; calcitonin receptorlike receptor; transforming growth factor-beta Adrenomedullin (ADM) is a peptide that was originally isolated from human pheochromocytoma tissues.1 It consists of 52 amino acids, and shares homology with the calcitonin gene-related peptide (CGRP) and amylin.1 ADM derives from the post-translational proteolytic cleavage of pro-ADM, the last step of which involves the conversion of inactive ADM to active ADM by the peptidyl-glycine alpha-amidating monooxygenase. ADM acts by binding calcitonin receptor-like receptor (CRLR), whose interaction with the subtypes 2 and 3 of a family of receptor activitymodifying proteins (RAMP) gives rise to 2 distinct ADM receptors, named ADM1 (CRLR/RAMP2) and ADM2 (CRLR/ RAMP3) receptors, whereas interaction of RAMP1 with CRLR leads to the formation of the so-called calcitonin gene-related peptide receptor. 2-4High levels of ADM have been found in the human adrenal medulla, heart, lung, kidney and pancreas, as well as in pheochromocytoma tissues. 1,5 In addition, cultured endothelial cells (ECs) and vascular smooth muscle cells of different species have been shown to produce and secrete ADM.6 ADM is multifunctional in nature, regulating the proliferation, differentiation and migration of a number of different cell lines...

Hypoxia-inducible proto-oncogene Pim-1 is a prognostic marker in pancreatic ductal adenocarcinoma

“…Immunocytochemistry was performed with minor modifications of a previously described protocol. 27 Aspc-1 cells were seeded at 80% and 20% confluency on sterile Teflon-coated slides (ER-208B-AD-CE24, Erie Scientific Company, Portsmouth, NH). Twenty-four hours later, the existing medium was changed to 0.1% and 10% FCS medium and incubated at 37°C for another 24 h. Cells were then washed with PBS and fixed in 4% paraformaldehyde (10 min), incubated in 30 mM Glycine/PBS (5 min), and permeabilized with 0.1% TritonX100 (5 min).…”

Adrenomedullin Blockade Suppresses Growth of Human Hormone–Independent Prostate Tumor Xenograft in Mice

“…The ability of CLR/RAMP2 and CLR/RAMP3 to respond with high affinity to adrenomedullin implies the existence of two molecularly distinct adrenomedullin receptors referred to as AM 1 and AM 2 receptors, respectively (9). Adrenomedullin is widely expressed in a variety of tumor types (10) and was shown to be mitogenic for many human cancer cell lines in vitro (4,(11)(12)(13)(14)(15). Several in vivo studies have shown a reduction of tumor angiogenesis and growth upon the treatment with neutralizing adrenomedullin antibodies (13), adrenomedullin receptor antagonist (16,17), or adrenomedullin receptor interference (14).…”

Expression and role of adrenomedullin in renal tumors and value of its mRNA levels as prognostic factor in clear‐cell renal carcinoma

“…AM activates many transduction pathways such as PI3-kinase/AKT, FAKinase and MAPKinase pathways in endothelial cells, 14 raf-ras pathway in breast cancer cell lines, 15 and cGMP-protein kinase G in prostate cancer LNCaP cell line. 16 AM is highly expressed in a variety of malignant tissues such as glioblastoma, 5 pancreatic carcinomas, 17 and cRCC. 18 In kidney, AM is found in a few distal tubules, collecting ducts and urinary tract.…”