Receptor in Human Pancreatic Adenocarcinomas Aberrant Expression of Type I Fibroblast Growth Factor

Acidic and basic fibroblast growth factors are mitogenic polypeptides that are overexpressed in pancreatic cancer. To determine whether fibro blast growth factors may exert direct effects on pancreatic cancer cells in vivo, we compared the expression of the high-affinity type I fibroblast growth factor receptor (FGFR-1) in human pancreatic tissues. In the normal pancreas, FGFR-1 immunostaining was seen mainly in acinar cells. In pancreatic cancers, FGFR-1 was abundant in duel al-like cancer cells which also exhibited many FGFR-1 MIUNA in situ hybridization grains. Analysis by the polymerase chain reaction and RNase protection revealed that the 2-immunoglobulin-like and the 3-immunoglobulin-like forms of FGFR-1 were expressed in all tissue samples, and that the 2-im munoglobulin-like form was overexpressed in the cancer tissues by com parison with the normal tissues. These findings suggest that the 2-immu noglobulin-like form of FGFR-1 may contribute to aberrant autocrine and paracrine pathways in pancreatic cancer. Introduction The FGF3 family consists of a number of homologous polypeptide growth factors that have an affinity for heparin and glycosaminoglycans and that participate in the regulation of biological processes in numerous cell types (1-4). This family currently includes aFGF, bFGF, keratinocyte growth factor, FGF-4 (Kaposi FGF), FGF-5, FGF-6, the gene product of int-2, androgen-induced growth factor, and FGF-9 (1-6). FGFs are mitogenic, promote angiogenesis and chemotaxis, and participate in the regulation of cellular differentiation and tissue repair. aFGF and bFGF are overexpressed in a significant proportion of pancreatic cancers,4 raising the possibility that FGFs may exert autocrine and paracrine effects in these cancers. It is not known, however, whether FGF receptors are expressed in these tu mors. To date, five distinct high-affinity FGF receptors, designated as FGFR-1, -2, -3, -4, and -5 (the latter is also known asflg-2) have been cloned and sequenced (7). All five receptors contain an intracellular domain that possesses intrinsic tyrosine kinase activity but which is separated into two contiguous regions, and an extracellular domain that has 3 immunoglobulin-like (3-Ig) regions (7). As a result of alternative splicing, in some cell types FGFR-1 and -2 exhibit a number of variants, including the loss of the first immunoglobulin region and the generation of a 2-Ig form (7). In the present study, we used immunohistochemical and in situ hybridization techniques to examine FGFR-1 localization and expression in normal and malignant Received 7/16/93; accepted 9/2/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by USPHS Grant DK-44948 awarded by the NIH to M. K. 2 To whom requests for reprints should be addressed, at Division of Endocrinology and Metabolism, Medical Sciences I, C240, University of California, Irvine, CA92717. -1The abbreviations used are: FGF, fibroblast growth factor; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; FGFR, fibroblast growth factor receptor; EOF, epidermal growth factor; cDNA, complementary DNA; PCR, polymerase chain reaction; 2-Ig, 2-immunoglobulin-like; 3-Ig, 3-immunoglobulin-like. 4 Y. Yamanaka, H. Friess, M. Buchler, H. G. Beger, E. Uchida, M. Onda, M. S. Kobrin, and M. Korc. Overexpression of acidic and basic fibroblast growth factors in human pancreatic cancer correlates with advanced tumor stage, submitted for publication. human pancreatic tissues. We also used a PCR-based strategy and a sensitive RNase protection assay to determine whether there is dif ferential expression of the 3and 2-Ig forms of FGFR-1 in the normal and cancerous tissues. We now report that human pancreatic adenocarcinomas express high levels of the 2-Ig form of FGFR-1. Materials and Methods Monoclonal anti-FGFR-1 antibody, previously shown to be useful for im munohistochemical analysis (8), was purchased from Chemicon Int., Inc. (Temecula, CA); biotinylated goat IgG from Kirkegaard & Perry Laboratories (Gaithersburg, MD); GeneScreen membranes from New England Nuclear (Boston, MA); pBluescript IISK+ vector from Stratagene, LaJolla, CA; pGEM3Zf vector from Promega Biotech., Madison, WI); Superscript reverse transcriptase from BRL, Gaithersburg, MD; Taq polymerase from PerkinElmer Cetus Corp., Norwalk, CT; intensifying screens from Eastman Kodak Co. (Rochester, NY); [a-32P]dCTP (3000 Ci/mmol) and [a-35S]UTP (1000 Ci/mmol) from Amersham, Inc. (Arlington Heights, IL). Human FGFR-1 pCDllS cDNA (9) was a gift from Dr. M. Jaye, Rorer Central Research, Inc. (King of Prussia, PA). Pancreatic cancer tissues were obtained from 5 patients (2 female, 3 male) undergoing pancreatic cancer surgery. Normal pancreatic tissues were obtained from 5 individuals (2 female, 3 male) through an organ donor program. Immediately following surgical removal, all tissue samples were either fixed in Bouin's solution or frozen in liquid nitrogen. The tumor samples were classi fied as pancreatic ductal adenocarcinomas according to the TNM classification for pancreatic tumors (10). All studies were approved by the Human Subjects Committee of the University of California, Irvine, CA. Immunohistochemistry. Paraffin-embedded tissue sections were subjected to immunostaining by using a streptavidin-peroxidase method. After blocking endogenous peroxidase activity with 0.03% hydrogen peroxide in methanol, the sections were incubated for 40 min at 23°Cwith 10% normal horse serum and overnight at 4°Cwith anti-FGFR-1 (1:50 dilution) antibodies (11). Bound antibody was detected with a biotinylated anti-mouse IgG secondary antibody and streptavidin-peroxidase complex, using diaminobenzidine tetrahydrochloride as the substrate. Counterstaining was performed with Mayer's hematoxylin. Incubation of sections with nonimmunized mouse IgG instead of a primary antibody failed to reveal any immunostaining. In Situ Hybridization. Pancreatic tissue sections were deparaffinized, treated with 1 fAg/ml proteinase K, incubated for 3 h at 42°Cin hybridization solution, and hybridized overnight at 50°C,using 200,000 cpm of the FGFR-1 riboprobe and 50 /j.g of yeast tRNA (11). The sections were then digested with RNase A, washed, dehydrated, coated with NTB2 autoradiography emulsion, and exposed for 6 days. After development the slides were counterstained with Mayer's hematoxylin. Pretreatment of the slides with excess RNase abolished the hybridization signal. The antisense riboprobe consisted of a 435-base pair EcoRl/Pstl fragment of the human FGFR-1 cDNA (9) which was subcloned into the pGEM3Zf vector and labeled with [a-35S]UTP (11). The correspond ing sense probe failed to produce a signal. PCR Analysis. Oligonucleotide primers were synthesized on an Applied Biosystem 391 DNA synthesizer and purified by electrophoresis (12). Two primer sequences (EC1 and EC2) were used to amplify the extracellular domain of FGFR-1 (Fig. 1): EC1 S'-CGCTCTAGAGCAGAACTGGGATGTGGGGCTG-S' EC2 S'-CTCGGATCCAGGGCTTCCAGAACGGTC-S' cDNAs were synthesized from total RNA (2 jug/sample) isolated from 5 human