Nakayama T, Yoshizaki A, Naito S, Wen CY, Alipov G, Yakata Y, Sekine I. Expression of Ets-1 proto-oncoprotein in gastrointestinal stromal tumors, leiomyomas and schwannomas. World J Gastroenterol 2006; 12(11): 1743-1746
Corresponding Author of This Article
Toshiyuki Nakayama, MD, Department of Molecular Pathology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. firstname.lastname@example.org
Article-Type of This Article
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Expression of Ets-1 proto-oncoprotein in gastrointestinal stromal tumors, leiomyomas and schwannomas
Toshiyuki Nakayama, Ayumi Yoshizaki, Shinji Naito, Chun Yang Wen, Gabit Alipov, Yuichi Yakata, Ichiro Sekine
Toshiyuki Nakayama, Ayumi Yoshizaki, Chun Yang Wen, Yuichi Yakata, Ichiro Sekine, Department of Molecular Pathology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
Shinji Naito, Division of Pathology, Research Laboratory, National Ureshino Medical Center, Saga 843-0301, Japan
Chun Yang Wen, Department of Digestive Disease, Nanjing Drum Tower Hospital, Medical School of Nanjing University, Nanjing 210008, Jiangsu Province, China
Gabit Alipov, Tissue and Histopathology Section, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan
ORCID number: $[AuthorORCIDs]
Correspondence to: Toshiyuki Nakayama, MD, Department of Molecular Pathology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. email@example.com
Telephone: +81-95-8497107 Fax: +81-95-8497108
Received: October 11, 2005 Revised: October 25, 2005 Accepted: November 10, 2005 Published online: March 21, 2006
AIM: Gastrointestinal stromal tumors (GISTs) are rare. GISTs differ from other mesenchymal tumors of the gastrointestinal tract (e.g. leiomyomas and schwannomas). The purpose of this study was to investigate the role of Ets-1 in the growth and differentiation of GISTs.
METHODS: Twenty-eight GISTs, nine leiomyomas and six schwannomas were examined by immunohistochemical staining method for Ets-1 in this study. Specimens were selected from surgical pathology archival tissues at Nagasaki University Hospital.
RESULTS: Ets-1 protein was expressed in the cytoplasm of cells in all of these tumors. Immunohistochemical staining revealed that 27 GISTs (96.4 %), six leiomyomas (66.7 %), and five schwannomas (83.3 %) were positive for Ets-1. Ets-1 expression was statistically different between GISTs and leiomyomas (P < 0.005). However, there was no correlation between Ets-1 expression and clinical risk categories.
CONCLUSION: Ets-1 plays an important role in the growth and differentiation of GISTs, leiomyomas and schwannomas.
Citation: Nakayama T, Yoshizaki A, Naito S, Wen CY, Alipov G, Yakata Y, Sekine I. Expression of Ets-1 proto-oncoprotein in gastrointestinal stromal tumors, leiomyomas and schwannomas. World J Gastroenterol 2006; 12(11): 1743-1746
Gastrointestinal stromal tumors (GISTs) are rare mes-enchymal tumors of the gastrointestinal tract that may occur from the oesophagus to the anus, including the omentum. These tumors have a wide clinical spectrum from benign, incidentally detected nodules to frankly malignant tumors. Small GISTs are often detected incidentally during surgery for other conditions, during gastroscopy, or on routine X-ray[1,2]. GISTs may present with bleeding, perforation, pain, obstruction or a com-bination of these symptoms[3-6]. The mechanisms of tumorigenesis, progression and differentiation of GISTs are unknown. Traditionally, all primary mesnchymal spindle cell tumors of the gastrointestinal (GI) tract were uniformly classified as smooth muscle tumors (e.g., leiomyomas, cellular leiomyomas or leiomyosarcomas). Tumors with epithelioid cytologic features were design-ated leiomyoblastomas or epithelioid leiomyosarcomas. Recently, Sircar et al postulated that GISTs origin-ate from Cajal cells in the gastrointestinal tract and differ from leiomyomas and schwannomas, which are of mesenchymal cell origin. Cajal cells are thought to be gastrointestinal pacemaker cells that regulate intestinal motility. GISTs are characterized by frequent expression of the bone marrow leukocytic progenitor cell antigen CD34 and the c-kit proto-oncogene[8,11,12]. Ets-1 was characterized originally as the v-ets retroviral gene, one of two oncogenes (v-myb and v-ets) of the avian leukemia retrovirus, E26. The ets family of genes encodes transcription factors for mesodermal cell development during embryogenesis[14,15].
Ets-1 plays a role in the regulation of physiological processes such as cell proliferation and differentiation. Ets-1 also is expressed in astrocytes and vascular smooth muscle cells[17,18] but its expression has not been reported in Cajal cells. Increased Ets-1 expression was observed in several tumors in our previous studies[19-22]. We reported that Ets-1 is correlated with the progression of carcinoma cells of the stomach, pancreas, and thyroid and cells of astrocytic tumors[19-22]. These studies suggest that Ets-1 is involved in tumor growth and differentiation. However, there are no data concerning Ets-1 expression in GISTs, leiomyomas or schwannomas or the role of Ets-1 in the etiology of these tumors. The purpose of this study was to investigate the expression of Ets-1 in GISTs.
MATERIALS AND METHODS
A total of twenty-eight GISTs included 24 cases from the stomach and four from the small intestine. Nine leiomyomas included four from the oesophagus, two from the stomach and three from the large intestine, and five schwannomas included four from the stomach and one from the large intestine. Specimens were selected from surgical pathology archival tissues at Nagasaki University Hospital between 1999 and 2004. The GISTs were 0.8 - 12.0 cm in diameter, the leiomyomas were 0.1 - 4.5 cm, and the schwannomas were 0.6 - 5.0 cm. In this study, GISTs were defined as expressing both c-kit and CD34 surface antigens. GISTs were classified by risk categories, mitosis counts and tumor size. The number of mitoses was determined by counting 50 high-power fields (HPF, x400) in Nikon (Tokyo, Japan) E400 microscope. Leiomyomas were defined as expressing α-smooth muscle actin (SMA) but not c-kit, CD34 and S100-protein. Schwannomas were defined as expressing S100-protein but not c-kit, CD34 and SMA. Tumor identification/classification was determined by two independent pathologists (T. Nakayama and I. Sekine).
The subcellular localization of Ets-1 was determined in GISTs using a monoclonal antibody directed against the unique middle sequence of Ets-1 and this antibody was devoid of any cross-reaction with other proteins in the Ets family. Formalin-fixed and paraffin-embedded specimens were cut into 4 μm thick sections, deparaffinized and preincubated with normal bovine serum to prevent non-specific binding. The sections were incubated overnight at 4°C with the primary monoclonal antibody to human Ets-1 (1 g/L; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and then with a horseraddish peroxidase conjugated goat anti-mouse IgG antibody (0.4 g/L; Santa Cruz Biotechnology, Santa Cruz, CA). The reaction products were resolved using diaminobenzidine (DAB; DAKO Ltd., Glostrap, Denmark). Negative controls involved replacing the primary antibody with non-immunized mouse serum and human gastric cancer tissue served as the positive control. Ets-1 expression was classified into three categories depending upon the percentage of cells stained and/or the intensity of staining: -, 0% to 10 % tumor cells positive; +, 10% to 50 % tumor cells positive; and ++, > 50 % tumor cells positive.
The Stat View II program (Abacus Concepts, Inc., Berkeley, CA) was used for statistical analyses. Analyses comparing the degree of Ets-1 expression in GISTs, leiomyomas and schwannomas were performed using the Mann-Whitney’s test.
The results of immunohistochemical staining for Ets-1 are summarized in the Table 1. Ets-1 expression was heterogeneous in GISTs and localized to the cytoplasm (Figure 1). Twenty-three of the GISTs (82.1%) were strongly positive, four (14.3 %) were positive and one (3.6 %) was negative for Ets-1. Similarly, four of the schwannomas (66.7 %) were strongly positive, one (16.7 %) was positive and one (16.7%) was negative. However, only three of the leiomyomas (33.3 %) were strongly positive, three (33.3 %) were positive and three (33.3 %) were negative. There is a statistical difference in Ets-1 expression between the GISTs and the leiomyomas (P < 0.005). Positively stained cells, i.e. those classified as ++ or +, were found in 96.4 % of the GISTs, 66.7 % of the leiomyomas and 83.3 % of the schwannomas. Ets-1 was expressed in the cytoplasm of cells in all three tumors. However, normal stromal cells and smooth muscle cells showed faint or focal positivity of expression. GISTs were classified by risk categories, mitosis counts and tumor size in Table 2. In risk categories, all nine cases of high and intermediate groups were strongly expressed Ets-1 protein. In mitosis counts, all eight cases with over 6 mitoses per 50 HPF strongly expressed Ets-1. In tumor size, all five cases with over 5 cm strongly expressed Ets-1. However, there was no correlation between Ets-1 expression and each classification.
Table 1 Ets-1 immunohistochemistry in intestinal stromal tumors. n (%).
Figure 1 Ets-1 expression in GISTs (A-C), Leiomyomas (D) and Schwannomas (E).
(magnification; A, B:x20, C: x 200, D, E: x100).
Table 2 Ets-1 immunohistochemistry and risk categories in GIST. n (%).
Mitosis counts (per 50 HPF)
Tumour size (cm)
GISTs are known to originate from the Cajal cells of the neural crest and schwannomas are thought to originate from the peripheral nerve sheath cell. In this study, Ets-1 expression was higher in GISTs and schwannomas than in leiomyomas. Ets-1 expression has been reported in neural cells and astrocytes, but not yet in Cajal cells, cells that are all of neurons origin. Vascular smooth muscle cells also express Ets-1. These findings suggest that Ets-1 may play a role in neural differentiation of intestinal stromal tumors. Previous studies have demonstrated Ets-1 expression in several tumors and normal stromal cells[19-22,25]. Furthermore, Ets-1 has been shown to play a role in the proliferation and/or differentiation of stromal cells. We have shown already that Ets-1 may function as a growth factor in several tumors[19-22]. However, there have been no studies of Ets-1 expression in GISTs, leiomyomas and schwannomas, or of the potential role of Ets-1 in the growth of these tumors. Our results demonstrate substantial levels of Ets-1 expression in the cytoplasm of GIST, leiomyoma and schwannoma cells. These results suggest that Ets-1 may play a role in the growth and/or differentiation of intestinal tumors.
Ets-1 regulates the expression of many proteins, such as matrix metalloproteinases, urokinase type-plasminogen activator and parathyroid hormone-related peptide (PTHrP), which promote tumor growth and/or progression[26,27]. In our previous study, PTHrP and its receptor were found to be highly expressed in GISTs, leiomyomas and schwannomas. Ets-1 may promote tumor growth and/or progression through regulating the expression of these proteins.
In recent studies, mutations affecting c-kit that cause constitutive tyrosine kinase activation have been shown to be important for the pathogenesis of GIST[29,30]. Joensuu
et al reported a patient in whom STI-571 (imatinib, Gleevec), a tyrosine kinase inhibitor, was effective against a GIST. And STI-571 has proven to be remarkably efficacious in heavily pretreated GISTs patients with advanced disease in phase II clinical trials. The expression of the Ets family protein is upregulated by the activation of tyrosine kinase through the mitogen-activated protein kinase pathway. Ets-1 expression may be upregulated by the c-kit/tyrosine kinase pathway.
We are grateful to Mr. Toshiyuki Kawada (Nagasaki Uni-versity Graduate School of Biomedical Sciences) for his excellent immunohistochemical assistance.
S- Editor Wang J L- Editor Zhang JZ E- Editor Wu M
Miettinen M, Lasota J. Gastrointestinal stromal tumors-- definition, clinical, histological, immunohistochemical, and molecular genetic features and differential diagnosis.Virchows Arch. 2001;438:1-12.
Miettinen M, Sarlomo-Rikala M, Sobin LH, Lasota J. Esophageal stromal tumors: a clinicopathologic, immunohistochemical，and molecular genetic study of 17 cases and comparison with esophageal leiomyomas and leiomyosarcomas.Am J Surg Pathol. 2000;24:211-222.
Miettinen M, Sarlomo-Rikala M, Sobin LH, Lasota J. Gastrointestinal stromal tumors and leiomyosarcomas in the colon: a clinicopathologic, immunohistochemical, and molecular genetic study of 44 cases.Am J Surg Pathol. 2000;24:1339-1352.
Ranchod M, Kempson RL. Smooth muscle tumors of the gastrointestinal tract and retroperitoneum: a pathologic analysis of 100 cases.Cancer. 1977;39:255-262.
Shiu MH, Farr GH, Papachristou DN, Hajdu SI. Myosarcomas of the stomach: natural history, prognostic factors and management.Cancer. 1982;49:177-187.
Ueyama T, Guo KJ, Hashimoto H, Daimaru Y, Enjoji M. A clinicopathologic and immunohistochemical study of gastrointestinal stromal tumors.Cancer. 1992;69:947-955.
Appelman HD. Mesenchymal tumors of the gut: historical perspectives, new approaches, new results, and does it make any difference.Monogr Pathol. 1990;220-246.
Sircar K, Hewlett BR, Huizinga JD, Chorneyko K, Berezin I, Riddell RH. Interstitial cells of Cajal as precursors of gastrointestinal stromal tumors.Am J Surg Pathol. 1999;23:377-389.
Sanders KM. A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract.Gastroenterology. 1996;111:492-515.
Miettinen M, Virolainen M, Maarit-Sarlomo-Rikala . Gastrointestinal stromal tumors--value of CD34 antigen in their identification and separation from true leiomyomas and schwannomas.Am J Surg Pathol. 1995;19:207-216.
Kindblom LG, Remotti HE, Aldenborg F, Meis-Kindblom JM. Gastrointestinal pacemaker cell tumor (GIPACT): gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal.Am J Pathol. 1998;152:1259-1269.
Sarlomo-Rikala M, Kovatich AJ, Barusevicius A, Miettinen M. CD117: a sensitive marker for gastrointestinal stromal tumors that is more specific than CD34.Mod Pathol. 1998;11:728-734.
Leprince D, Gegonne A, Coll J, de Taisne C, Schneeberger A, Lagrou C, Stehelin D. A putative second cell-derived oncogene of the avian leukaemia retrovirus E26.Nature. 1983;306:395-397.
Kola I, Brookes S, Green AR, Garber R, Tymms M, Papas TS, Seth A. The Ets1 transcription factor is widely expressed during murine embryo development and is associated with mesodermal cells involved in morphogenetic processes such as organ formation.Proc Natl Acad Sci U S A. 1993;90:7588-7592.
Vandenbunder B, Pardanaud L, Jaffredo T, Mirabel MA, Stehelin D. Complementary patterns of expression of c-ets 1, c-myb and c-myc in the blood-forming system of the chick embryo.Development. 1989;107:265-274.
Lewin B. Oncogenic conversion by regulatory changes in transcription factors.Cell. 1991;64:303-312.
Fleischman LF, Holtzclaw L, Russell JT, Mavrothalassitis G, Fisher RJ. ets-1 in astrocytes: expression and transmitter-evoked phosphorylation.Mol Cell Biol. 1995;15:925-931.
Naito S, Shimizu S, Maeda S, Wang J, Paul R, Fagin JA. Ets-1 is an early response gene activated by ET-1 and PDGF-BB in vascular smooth muscle cells.Am J Physiol. 1998;274:C472-C480.
Nakayama T, Ito M, Ohtsuru A, Naito S, Nakashima M, Fagin JA, Yamashita S, Sekine I. Expression of the Ets-1 proto-oncogene in human gastric carcinoma: correlation with tumor invasion.Am J Pathol. 1996;149:1931-1939.
Ito T, Nakayama T, Ito M, Naito S, Kanematsu T, Sekine I. Expression of the ets-1 proto-oncogene in human pancreatic carcinoma.Mod Pathol. 1998;11:209-215.
Nakayama T, Ito M, Ohtsuru A, Naito S, Nakashima M, Sekine I. Expression of the ets-1 proto-oncogene in human thyroid tumor.Mod Pathol. 1999;12:61-68.
Kitange G, Kishikawa M, Nakayama T, Naito S, Iseki M, Shibata S. Expression of the Ets-1 proto-oncogene correlates with malignant potential in human astrocytic tumors.Mod Pathol. 1999;12:618-626.
Dittmer J, Gégonne A, Gitlin SD, Ghysdael J, Brady JN. Regulation of parathyroid hormone-related protein (PTHrP) gene expression. Sp1 binds through an inverted CACCC motif and regulates promoter activity in cooperation with Ets1.J Biol Chem. 1994;269:21428-21434.
Yoshizaki A, Nakayama T, Naito S, Sekine I. Expressions of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP-receptor (PTH/PTHrP-R) in gastrointestinal stromal tumors (GISTs), leiomyomas and schwannomas.Scand J Gastroenterol. 2004;39:133-137.
Plaat BE, Hollema H, Molenaar WM, Torn Broers GH, Pijpe J, Mastik MF, Hoekstra HJ, van den Berg E, Scheper RJ, van der Graaf WT. Soft tissue leiomyosarcomas and malignant gastrointestinal stromal tumors: differences in clinical outcome and expression of multidrug resistance proteins.J Clin Oncol. 2000;18:3211-3220.
Van Glabbeke M, van Oosterom AT, Oosterhuis JW, Mouridsen H, Crowther D, Somers R, Verweij J, Santoro A, Buesa J, Tursz T. Prognostic factors for the outcome of chemotherapy in advanced soft tissue sarcoma: an analysis of 2,185 patients treated with anthracycline-containing first-line regimens--a European Organization for Research and Treatment of Cancer Soft Tissue and Bone Sarcoma Group Study.J Clin Oncol. 1999;17:150-157.
Joensuu H, Roberts PJ, Sarlomo-Rikala M, Andersson LC, Tervahartiala P, Tuveson D, Silberman S, Capdeville R, Dimitrijevic S, Druker B. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor.N Engl J Med. 2001;344:1052-1056.
Conrad KE, Oberwetter JM, Vaillancourt R, Johnson GL, Gutierrez-Hartmann A. Identification of the functional components of the Ras signaling pathway regulating pituitary cell-specific gene expression.Mol Cell Biol. 1994;14:1553-1565.