The G691S RET Polymorphism Increases Glial Cell Line–Derived Neurotrophic Factor–Induced Pancreatic Cancer Cell Invasion by Amplifying Mitogen-Activated Protein Kinase Signaling

Hirozumi Sawai,Howard A Reber,Joseph Kim,Guido Eibl,Oscar J Hines,Dave S.B Hoon,Sascha Hasan,Yuji Okada,Kevork Kazanjian

doi:10.1158/0008-5472.can-05-2843

Hirozumi Sawai, Howard A Reber + Show 7 more

Open Access

https://doi.org/10.1158/0008-5472.can-05-2843

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Abstract

Mutations of the RET proto-oncogene are responsible for several inherited human diseases and may function as genetic modifiers of the disease. However, the role of RET mutations in pancreatic cancer has not been studied. Expression of the glial cell line-derived neurotrophic factor (GDNF) receptors RET and GDNF family receptor alpha1 (GFRalpha1) in human pancreatic cancer cells was determined by Western blot, immunofluorescence, and flow cytometry. The effect of GDNF on cell proliferation and invasion was assessed. Small interfering RNA and antibodies were used to evaluate the involvement of RET. The G691S RET polymorphism was analyzed by sequencing and restriction analysis. The modifying effect of G691S RET on GDNF-induced invasion and mitogen-activated protein kinase (MAPK) signaling was evaluated. Transfection studies with wild-type and mutated RET determined the functional role of the G691S polymorphism. Pancreatic cancer specimens and matched tissues were analyzed for the presence of the G691S RET polymorphism. GDNF receptors were found on all cell lines. GDNF increased pancreatic cancer cell proliferation and invasion, which was mediated by RET. The effect of GDNF was more profound in cells with the G691S RET polymorphism (P < 0.01). G691S RET correlated with an enhanced activation of the downstream extracellular signal-regulated kinase pathway. Overexpression of G691S RET increased pancreatic cancer cell invasion. The G691S RET polymorphism was also detected in human pancreatic tumors and represented a somatic mutation in some patients. These findings indicate that the G691S RET single nucleotide polymorphism may directly correlate with the aggressive growth of pancreatic cancers and may function as a genetic modifier or even low-penetrance gene.

Highlights

Pancreatic ductal adenocarcinoma is an extremely lethal disease, which is characterized by its propensity to infiltrate adjacent tissues and to metastasize even at early stages [1]
Six human pancreatic cancer cell lines were initially analyzed for the presence of the glial cell line–derived neurotrophic factor (GDNF) receptors
Our study clearly shows that GDNF increases proliferation and invasion of pancreatic cancer cells

Summary

Introduction

Pancreatic ductal adenocarcinoma is an extremely lethal disease, which is characterized by its propensity to infiltrate adjacent tissues and to metastasize even at early stages [1]. A characteristic pattern of genetic alterations in pancreatic cancer has been elucidated, which include activating mutations in oncogenes, e.g., KRAS, and loss-of-function mutations in tumor suppressor genes, e.g., INK4A and TP53. The genetic changes seem to temporally correlate with the development of pancreatic intraepithelial neoplasias, precursor lesions of pancreatic adenocarcinomas [1, 3]. It is still not well understood how these signature genetic lesions contribute to the biological characteristics of this disease. In addition to alterations in oncogenes and tumor suppressor genes, amplified autocrine and paracrine growth factor signaling loops, e.g., the epidermal growth factor pathway, contribute to the aggressive growth pattern of pancreatic cancers [4]. Germline genetic variants of these growth factors and their cognate receptors are being increasingly recognized as critical modulators of cancer biology [5]

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