Abstract 3023: Decreased neutral endopeptidase expression regulates the autocrine proliferative effects of substance P on cholangiocarcinoma growth

Abstract

Abstract Cholangiocarcinoma (CCA) originates from the neoplastic transformation of cholangiocytes, which are the epithelial cells that line the bile ducts. CCAs are neuroendocrine tumors and secrete and respond to neuropeptides with alterations in cell proliferation via autocrine pathways. For example, the expression/secretion of neuroendocrine factors such as NPY and serotonin are dysregulated in cholangiocarcinoma, which exert local control on tumor cell proliferation. The neuropeptide substance P (SP) stimulates normal biliary hyperplasia by binding to the neurokinin-1 receptor (NK-1R) stimulating ERK1/2 activity. Neutral endopeptidase (NEP) is a metalloprotease that degrades a number of secreted neuropeptides including SP. The AIM of our study was to evaluate the role of NEP in the autocrine regulation of CCA growth by SP. METHODS: The intra- and extra-hepatic CCA lines, Mz-ChA-1, SG231, CCLP, HuCCT-1, HuH-28, and TFK-1, and non-malignant biliary line, H-69, were utilized. We evaluated NK1R, tachykinin 1 (TAC1, gene encoding SP) and NEP expression by qPCR, FACS and immunofluorescence in cell lines and by immunohistochemistry in commercially available CCA tissue arrays. The secretion of SP in all lines was measured by EIA. The dose-dependent effect of SP (10−6 to 10−11 M for 48 hrs) on cell growth and SP-induced (10−7 M) cell growth in the presence/absence of PD98059 (MEK1 inhibitor) was evaluated by MTS proliferation assay. The effect of SP on phosphorylation of ERK1/2 was determined by Western blot in the presence/absence of PD98059. To determine the role of NEP in the regulation of CCA growth, NEP was overexpressed in Mz-ChA-1 cells before evaluating: (i) NEP expression by qPCR and SP secretion by EIA kits; (ii) proliferation by MTS assay; and (iii) expression of Cyclin D1, PCNA and VEGF-A/C (factors regulating CCA growth). RESULTS: All CCA cell lines and human CCA tissue array samples were positive for NK-1R and TAC1. The CCA lines secreted higher levels of SP compared to H69. Correspondingly, NEP expression levels were significantly reduced in CCA lines compared to nonmalignant H69 cells and in human CCA tissue arrays compared to normal bile ducts. SP increased CCA growth (∼60% at 10−7 M), which was dependent upon ERK1/2 activity. Overexpression of NEP (∼80%, the SP degrading enzyme) in Mz-ChA-1 cells decreased SP secretion and significantly decreased cell growth, which was associated with decreased expression of Cyclin D1, PCNA and VEGF-A/C compared to control transfected Mz-ChA-1. CONCLUSIONS: SP is mitogenic for CCA regulating growth in an autocrine fashion that is associated with decreased NEP expression. Targeting the NEP/SP/NK1R axis may represent a novel therapeutic approach for cholangiocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3023. doi:1538-7445.AM2012-3023