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Serine Protease Inhibitor Kazal Type 1 Promotes Proliferation of Pancreatic Cancer Cells Through the Epidermal Growth Factor Receptor

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Serine Protease Inhibitor Kazal Type 1 Promotes Proliferation of Pancreatic Cancer Cells Through the Epidermal Growth Factor Receptor Published OnlineFirst September 8, 2009; DOI: 10.1158/1541-7786.MCR-08-0567 Serine Protease Inhibitor Kazal Type 1 Promotes Proliferation of Pancreatic Cancer Cells through the Epidermal Growth Factor Receptor Nobuyuki Ozaki,1,2 Masaki Ohmuraya,1,2,3 Masahiko Hirota,4 Satoshi Ida,1,2 Jun Wang,1 Hiroshi Takamori,2 Shigeki Higashiyama,5 Hideo Baba,2 and Ken-ichi Yamamura1 1Division of Developmental Genetics, Institute of Molecular Embryology and Genetics, 2Department of Gastroenterological Surgery, Graduate School of Medical Sciences, and 3Priority Organization for Innovation and Excellence, Kumamoto University, 4Department of Surgery, Kumamoto Regional Medical Center, Kumamoto, Japan; and 5Department of Biochemistry and Molecular Genetics Protein Network Laboratory, CEREM, Ehime University Graduate School of Medicine, Toon, Ehime, Japan Abstract coexpressed not only in the early stage of cancer, Serine protease inhibitor, Kazal type 1 (SPINK1) is PanIN-1A, but also in advanced stages. Taken together, expressed not only in normal human pancreatic acinar these results suggest that SPINK1 stimulates the cells but also in a variety of pancreatic ductal neoplasms. proliferation of pancreatic cancer cells through There are structural similarities between SPINK1 and the EGFR/mitogen-activated protein kinase cascade. epidermal growth factor (EGF). Hence, we hypothesized (Mol Cancer Res 2009;7(9):1572–81) that SPINK1 binds to EGF receptor (EGFR) to activate its downstream signaling. We first showed that SPINK1 induced proliferation of NIH 3T3 cells and pancreatic Introduction cancer cell lines. We showed that SPINK1 coprecipitated Serine protease inhibitor, Kazal type 1 (SPINK1; ref. 1), with EGFRin an immunoprecipitation experiment and which is also known as pancreatic secretory trypsin inhibitor that the binding affinity of SPINK1 to EGFRwas about and tumor-associated trypsin inhibitor, is mainly produced in half of that of EGF using quartz-crystal microbalance the acinar cells of the exocrine pancreas. Tumor-associated (QCM) technique. As expected, EGFRand its downstream trypsin inhibitor was initially isolated fromthe urine of a pa- molecules, signal transducer and activator of transcription tient with ovarian cancer (2) and was later found to be iden- 3, v-Akt murine thymoma viral oncogene homologue, tical to SPINK1 (3). The role of SPINK1 has been postulated and extracellular signal-regulated kinase 1/2, were to be the prevention of inadvertent proteolysis in the pancreas phosphorylated by SPINK1 as well as EGF. To determine caused by intra-acinar premature activation of trypsinogen which pathway is the most important for cell growth, (4-6). However, considerable amounts of SPINK1 were de- we further analyzed the effect of inhibitors. Growth tected in many extrapancreatic tissues, including the stomach, stimulation by EGF or SPINK1 was completely inhibited colon, small intestine, liver, lung, kidney, and ovary (7-10). In by EGFRand mitogen-activated protein kinase/ addition, Ogawa et al. (11, 12) found elevated serumSPINK1 extracellular signal-regulated kinase kinase inhibitor but concentrations in patients with severe systemic inflammation. not by Janus-activated kinase and phosphoinositide We also found that Spink3, a mouse homologue of human 3-kinase inhibitors. To further analyze the clinical SPINK1, was expressed in several tissues, including brain importance of SPINK1 in the development of pancreatic or urogenital system, during embryonic development (10). cancer, we examined the expression of SPINK1 and EGFR These results suggest that SPINK1 has additional functions in pancreatic tubular adenocarcinomas and pancreatic in many tissues other than the pancreas. intraepithelial neoplasm. Both SPNK1 and EGFRwere Various cancers, including pancreatic cancers, produce SPINK1 (8, 13-16). SPINK1 is also a marker of poor prognosis (3, 17-20). Quantitative RT-PCR identified SPINK1 as a gene associated with early recurrence of intrahepatic cholangiocarci- Received 12/15/08; revised 6/5/09; accepted 6/14/09; published OnlineFirst 9/8/09. noma after resection (19). Thus far, the expression of SPINK1 Grant support: KAKENHI (Grant-in-Aid for Scientific Research) in Priority Areas “Integrative Research Toward the Conquest of Cancer” and Grant-in-Aid in tumor cells has been explained by its coexpression with for Young Scientists (B) fromthe Ministry of Education, Culture, Sports, Science tumor-associated trypsin, which is thought to participate in and Technology of Japan, Osaka Foundation of Promotion of Clinical tumor-associated protease cascades mediating tumor invasion Immunology, and Pancreas Research Foundation of Japan. The costs of publication of this article were defrayed in part by the payment of (13). However, the role of SPINK1 in malignant tumors remains page charges. This article must therefore be hereby marked advertisement in to be elucidated (19). accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The possibility that SPINK1 was a growth factor was raised Requests for reprints: Ken-ichi Yamamura, Division of Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto when SPINK1 was shown to have some structural similarities University, 2-2-1 Honjo, Kumamoto 860-0811, Japan. Phone: 81-96-373-6596; to epidermal growth factor (EGF). They share ∼50% amino acid Fax: 81-96-373-6599. E-mail: [email protected] Copyright © 2009 American Association for Cancer Research. sequence homology (21, 22) and have similar numbers of amino doi:10.1158/1541-7786.MCR-08-0567 acid residues (56 and 53, respectively; molecular weights 1572 Mol Cancer Res 2009;7(9). September 2009 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst September 8, 2009; DOI: 10.1158/1541-7786.MCR-08-0567 Activation of EGFR andIts Downstream Targets by SPINK1 1573 ∼6 kDa) and three intrachain disulfide bridges (21-23). In addi- in AsPC-1 and MIAPaCa-2 cell lines. As shown in Fig. 2A, tion, SPINK1 has been shown to bind specifically to several cell SPINK1 was clearly detected in both cell lines, suggesting that lines (24), suggesting that SPINK1 works as a growth factor in SPINK1 did bind with EGFR. tumor cells. SPINK1 was shown to stimulate the growth of NIH We then examined the binding affinity of SPINK1 with 3T3 fibroblasts (25, 26), human endothelial cells (27), intestinal EGFR, HER2 (ErbB2), HER3 (ErbB3), or HER4 (ErbB4) epithelial cells (28), and rat pancreatic cancer cells (29). On the in comparison with EGF using a quartz-crystal microbalance other hand, the identity of the receptor for SPINK1 has been a (QCM) technique. In this method, qualitative and quantitative controversial matter. SPINK1 was shown to bind to a cell sur- changes resulting fromintermolecularinteractions can be face receptor, which is distinct fromthe EGF receptor (EGFR; monitored and frequency (Hz) decreases in proportion to the refs. 24, 30). In contrast, Fukuoka et al. (31) reported that change of molecular mass caused by binding of ligands, SPINK1 competed with mouse EGF for binding to EGFR on which are immobilized on the gold electrode surface, with re- NIH 3T3 fibroblasts. We showed previously that excessive au- ceptors. When the EGFR extracellular domain-IgG-Fc fusion tophagy (cellular self-digestion) is induced in pancreatic acinar protein was injected into the equilibrated solution containing cells deficient in Spink3 (5). Interestingly, residual duct-like a SPINK1-immobilized or EGF-immobilized sensor chip, cells in the tubular complexes of the pancreas showed no sign the frequency decreased to 244.3 or 421.8 Hz, respectively of acinar cell regeneration in spite of the expression of EGFR.6 (Fig. 2B), suggesting that the binding affinity of SPINK1 is Based on these findings, we hypothesized that SPINK1 is about half that of EGF. There was no change in frequency a ligand for the EGFR. To test this, we investigated the (a) when the neuregulin-1 (NRG1) sensor chip was used. NRG1 proliferative activity of SPINK1 using various cell lines, (b) is known to bind to HER3 and HER4 as a specific ligand. binding of SPINK1 to receptors including EGFR, (c) activation When the HER2 extracellular domain-IgG fusion protein was of a downstreamsignal cascade, and ( d) expression of SPINK1 injected into the equilibrated solution containing a SPINK1- and EGFR in pancreatic tubular adenocarcinomas and pancre- immobilized, EGF-immobilized, or NRG1-immobilized sensor atic intraepithelial neoplasm(PanIN). chip, no decrease of frequency occurred (Fig. 2C). When the HER3 extracellular domain-IgG fusion protein was injected into the equilibrated solution containing SPINK1- or EGF- Results immobilized sensor chip, the frequency did not decrease. Growth-Stimulating Activity of SPINK1 on NIH 3T3 However, the frequency decreased to 235.4 Hz when the Fibroblast andPancreatic Cancer Cell Lines NRG1-immobilized sensor chip was used (Fig. 2D). When To examine whether EGF or SPINK1 act as growth factors, the HER4 extracellular domain-IgG fusion protein was injected NIH 3T3 cells, four pancreatic cancer cell lines (AsPC-1, MIA- into the equilibrated solution containing SPINK1- or EGF- PaCa-2, PANC-1, and Capan-2), and one breast cancer cell line immobilized sensor chip, the frequency did not decrease. The (BT-474) were treated with EGF (100 pg/mL and 1, 10, and frequency decreased to 112.5 Hz when the NRG1-immobilized 100 ng/mL) or SPINK1 (1 and 10 ng/mL; Fig. 1). sensor chip was used
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