Expression of the Ets-1 Proto-Oncogene in Human Gastric Carcinoma Correlation with Tumor Invasion

American Journal ofPathology, Vol. 149, No. 6, December 1996 Copynight © American Society for Investigative Pathology

Toshiyuki Nakayama,* Masahiro Ito,* tide probe, also confirmed the presence ofEts-1 Akira Ohtsuru,t Shinji Naito,*t mRNA in gastric carcinomas. Expression ofEts-I Masahiro Nakashima,* mRNA was also detected in four different kinds James Alexander Fagin,t ofcultured human gastric carcinoma ceU lines by Shunichi Yamashita,t and Ichiro Sekine* the reverse transcription polymerase chain reac- From the Department ofPathology* and Department of Cell tion method. Thesefindings suggest that Ets-I is Physiology,t Atomic Disease Institute, Nagasaki University overexpressed in gastric mucosal ceUs that have School ofMedicine, Nagasaki, Japan, and the Division of undergone malignant conversion and that Ets-l Endocrinology and Metabolism,* Department ofInternal is one of thefactors involved in the penetration Medicine, University of Cincinnati, College ofMedicine, ofgastric carcinoma beyond the muscularis mu- Cincinnati, Ohio cosa. (AmJPathol 1996,149:1931-1939)

The occurrence and progression of cancer is sug- The proto-oncogene Ets-1 is a transcription fac- gested to be related to a series of genetic events tor known to control the expression ofa number affecting the structure and/or expression of a num- ofgenes involved in extracelular matrix remod- ber of oncogenes, and eling and has been postulated to play a role in anti-oncogenes, growth fac- ceU migration and tumor invasion. To elucidate tors. However, the mechanism of invasion of gastric the involvement ofEts-1 in human gastric carci- carcinomas has not been fully elucidated. Early car- nomas, we examined 11 cases of gastric ade- cinomas are subclassified into intramucosal and noma and 110 cases ofgastric carcinoma by im- submucosal types, depending on whether the carci- munohistochemistry and compared the degree noma has penetrated beyond the muscularis mu- ofEts-1 expression with the depth ofcarcinoma cosa. The submucosal cancers have relatively invasion. Ets-I was not expressed either in the higher rates of lymph duct and venous invasion and normal gastric epithelium or in gastric adeno- lymph node metastasis.1 However, the factors that mas. Among the 110 cases with gastric adenocar- cause the carcinoma to invade the submucosa in the cinoma, 70 (63.6%) showed positive stainingfor stomach have not been completely elucidated. the Ets-1 protein. In mucosal carcinomas, only 3 Ets-1 was originally characterized as the v-ets ret- of 26 cases (11.5%) showed positive immuno- roviral gene, one of the two oncogenes (v-myb and staining for Ets-1. In contrast, 67 of 84 cases v-ets) in the avian leukemia retrovirus E26.2 The Ets (79.8%) with submucosal or more invasive car- family of genes encode transcription factors for me- cinomas showed immunopositivity and intense sodermal cell development during the embryonal stainingfor Ets-l in the tumor ceUs. The pattern period.3'4 Ets-1 plays a role in the regulation of phys- ofEts-l immunostaining in mucosal carcinomas iological processes such as cell proliferation and was weak and differedfrom that of other local differentiation.5 Ets-1 is also associated with invasive invasive carcinomas (P < 0.001). Histologically, processes in the stromal tissues of human carcino- signet-ring ceU and mucinous carcinomas expressed relatively weakpositivityfor Ets-1 Ets-1. Accepted for publication August 12, 1996. expression correlated significantly with the Address reprint requests to Dr. Toshiyuki Nakayama, Department presence of lymph node metastasis (P < 0.001). of Pathology, Atomic Disease Institute, Nagasaki University School In situ hybridization, using an Ets-1 oligonucleo- of Medicine, 1-12-4 Sakamoto, Nagasaki 852, Japan.

1931 1932 Nakayama et al A/P December 1996, Vol. 149, No. 6 mas.6 However, the biological function of the Ets-1 MKN-1 (adenosquamous carcinoma), MKN-45 proto-oncogene remains unknown. The processes of (poorly differentiated adenocarcinoma), NUGC-1 tumor invasion and metastasis are thought to de- (adenocarcinoma), and KATO-111 (signet-ring cell pend on the increased proteolytic activity of the in- carcinoma). 15,16 vading tumor cells.7 Matrix metalloproteinases, cathepsins B and D, and plasminogen activator have Immunohistochemistry been proposed to participate in the metastatic cas- cade.7-12 Ets-1 protein interacts with the urokinase- Formalin-fixed and paraffin-embedded tissues were type plasminogen activator gene enhancer and with cut into 4-,tm sections, deparaffinized in xylene, and the promoters of the stromelysin-1 and collage- rehydrated in phosphate-buffered saline. Deparaf- nase-1 genes.6' 13 Ets-1, therefore, is suggested to finized sections were preincubated with normal bo- regulate increased tumour invasion by activating the vine serum to prevent nonspecific binding and then expression of urokinase-type plasminogen activator, incubated overnight at 40C with an optimal dilution stromelysin, and collagenase. Based on these find- (0.1 ,tg/ml) of a primary polyclonal antibody against ings, we hypothesized that Ets-1 may be involved in human Ets-1 (C-20, raised against the carboxyl-ter- the progression and invasion of gastric carcinomas. minal domain of the Ets-1 protein; Santa Cruz Bio- The purpose of the present study was to evaluate technology, Santa Cruz, CA).17 The slides were se- the role of Ets-1 in the progression of human gastric quentially incubated with an alkaline-phosphatase- adenocarcinoma, especially concerning penetration conjugated horse anti-rabbit immunoglobulin beyond the muscularis mucosa. antibody, and the reaction products were resolved using a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium chloride (BCIP/NBT; and Methods GIBCO/BRL, Gaithersburg, MD). Preabsorption of Materials the primary antibody with excess recombinant Ets-1 We studied 11 gastric adenomas and 110 primary peptide (Santa Cruz Biotechnology) was used as a human gastric adenocarcinomas including 26 mu- negative control. Adrenal gland tissue3 served as the cosal carcinomas, 32 submucosal infiltrative carci- internal positive control for Ets-1 immunostaining. nomas, 14 carcinomas invading proprial muscle lay- Analysis of the immunohistochemical staining was ers, 16 carcinomas reaching the subserosa, and 22 performed by two investigators (T. Nakayama and M. carcinomas penetrating the serosal surface. All tu- Ito). Ets-1 expression was classified into three cate- mor specimens were obtained from patients oper- gories depending on the percentage of cells stained ated at Nagasaki University Hospital between 1992 and/or the intensity of staining: -, 0 to 10% positive and 1994. Each tumor was assigned a histological cells; +, 10 to 50% positive tumor cells; and ++, type and a depth grading of infiltration according to >50% positive tumor cells. the Japanese Classification of Gastric Carcinoma by the Japanese Research Society for Gastric Can- In cer.14 Histologically, of the 110 primary human gas- Situ Hybridization tric adenocarcinomas, 13 were papillary adenocar- In situ hybridization for the detection of human Ets-1 cinomas, 26 were tubular adenocarcinomas of the mRNA was performed using an oligonucleotide well differentiated type, 27 were tubular adenocarci- probe complementary to a fragment of human Ets-1 nomas of the moderately differentiated type, 14 were mRNA.18 The sequence of the oligonucleotide probe poorly differentiated adenocarcinomas of the solid is 5'-GCCCAGCTTCATCACAGAGTCCTATCAGAC- type, 13 were poorly differentiated adenocarcinomas 3', and it does not cross-hybridize with other mRNA of the nonsolid type, 10 were signet-ring cell carci- sequences. The probe was labeled with 5'-tailed nomas, and 7 were mucinous adenocarcinomas. A digoxigenin (Greiner Japan, Tokyo, Japan) and pu- total of 15 specimens of normal gastric mucosal rified by high performance liquid chromatography. tissue, composed of tissue surrounding carcinoma Four cases of human gastric adenocarcinoma were cells, were evaluated as the normal control. Diagno- studied by in situ hybridization. In all of these cases, sis was established by two independent pathologists we were able to obtain relatively fresh (within 6 (T. Nakayama and M. Ito), and cases of questionable months) paraffin-embedded sections. The presence diagnosis were omitted from the study. Four clonal of cytoplasmic RNA was confirmed by using a cultured cell lines established from human gastric methyl green pyronine staining solution (Muto Pure carcinomas were used for the reverse transcriptase Chemicals, Tokyo, Japan). Prehybridization was car- polymerase chain reaction (RT-PCR) analysis: ried out as described previously.19 The sections Ets-1 Expression in Gastric Carcinoma 1933 AJP December 1996, Vol. 149, No. 6 were treated with 0.2 N HCI for 20 minutes and primers were 5'-GGGTGACGACTTCTTGTTTG-3' digested with 100 ,ug/ml proteinase K (Sigma Chem- (sense) and 5'-GTTAATGGAGTCAACCCAGC-3' ical Co., St. Louis, MO) for 10 minutes at 37°C. After (antisense). The human ,B-actin PCR primers were postfixation in 4% paraformaldehyde, each section 5'-TCCTCCCTGGAGAAGACTA-3' (sense) and 5'- was covered with 20 ,ul of denatured hybridization AGTACTTGCGCTCAGGAGGA-3' (antisense). The mixture containing 4% dextran sulfate, 125 ,ug/ml Ets-1 and 1-actin primers are predicted to amplify sonicated salmon sperm DNA, 9% deionized form- 274- and 313-bp DNA fragments, respectively. Both amide, 2.5 ,ug/mI yeast tRNA, 5X Denhardt's me- primer pairs were chosen to span introns of their dium, 1 mmol/L EDTA (pH 7.4), 0.6 mol/L NaCI, 10 respective human genes. Samples were subjected mmol/L Tris/HCI, and 1 ,ug/ml digoxigenin-labeled to 28 cycles of PCR amplification using a thermocy- Ets-1 oligonucleotide probe, and placed in a moist cler. Each cycle included denaturation at 940C for 1 chamber, where it was incubated at 370C for 15 minute, annealing at 570C for 1 minute, and primer hours. After washing, in situ detection was accom- extension at 72°C for 1.5 minutes. An aliquot of each plished with a digoxigenin detection kit (GIBCO/ amplification mixture was subjected to electrophore- BRL). Briefly, the slides were incubated with 100 jdl sis on a 2.0% agarose gel, and DNA was visualized of blocking solution for 15 minutes at room temper- by ethidium bromide staining. ature and incubated with the streptavidin-alkaline phosphatase complex. Ets-1 mRNA expression was evaluated by comparing alkaline phosphatase stain- Statistical Analysis ing using BCIP/NBT with the results obtained from The Stat View 11 program (Abacus Concepts, Berke- the positive and negative controls. Each slide was ley, CA) was used for statistical analyses. Analyses studied in duplicate, and negative controls were comparing the intensities of Ets-1 expression were made using the digoxigenin-labeled sense oligo- performed by Student's t-test. probe (5'-GTCTGATAGGACTCTGTGATGAAGCT- GGGC-3'). RNAse treatment was carried out before hybridization as another negative control. Slides of a human adrenal gland served as a positive control.3 Results The results from immunohistochemical staining are RT-PCR summarized in Table 1. Ets-1 antigen was expressed heterogeneously in carcinomas. Strong staining of Total RNA was prepared from the control human Ets-1 was observed in 63.6% of the carcinomas (70 gastric carcinoma cell lines MKN-1, MKN-45, of 1 10). Ets-1 immunoreactivity in relation to histolog- NUGC-1, and KATO-11115 16 that were provided by ical type is shown in Table 1. Poorly differentiated Dr. S. Yamashita (Nagasaki University) from normal adenocarcinomas of the solid and nonsolid type and gastric mucosa obtained from two patients with gas- tubular adenocarcinomas of the moderately differen- tric ulcer and from human adrenal tissue using the tiated type gastric carcinomas stained strongly for acid guanidine phenol method.20 Ets-1. However, well differentiated (papillary adeno- Four control human gastric carcinoma cell lines carcinomas and tubular adenocarcinomas of the were incubated at 37°C in a humidified atmosphere well differentiated type) and mucinous gastric carci- of 5% CO2 and 95% air. Cultures were maintained in nomas expressed relatively weak positivity of Ets-1. basal medium supplemented with 10% fetal calf se- In signet-ring cell carcinomas, most cases were neg- rum (GIBCO/BRL). Plastic culture dishes were pur- ative for Ets-1 (2 of 10, 20.0%), and all 1 1 cases of chased from Becton Dickinson (Oxnard, CA). adenoma and all 15 cases of normal mucosa were Cellular RNA (1 ,ug) was incubated at 37°C for 1 negative for Ets-1. hour in 50 ,ul of reverse transcriptase buffer contain- Figure 1 shows a representative example of strong ing 20 U of RNAsin (Promega Corp., Madison, WI), immunohistochemical Ets-1 staining in a carcinoma 100 pmol of random hexamer primers (Boehringer invading the subserosa. Ets-1 protein was detected Mannheim, Mannheim, Germany), and 400 U of in both the cytoplasm and the nucleus of almost all Moloney murine leukemia virus reverse transcriptase carcinomas (Figure 1 B). In one case, Ets-1 protein (GIBCO/BRL). Reverse transcription was terminated was predominantly located in the nucleus (Figure by heating at 950C for 10 minutes, and 20% of the 1C), but even in this case, Ets-1 was also partially resulting cDNA was removed for PCR. PCR samples expressed in the cytoplasm (Figure 1C, arrow- were incubated with 50 pmol of each primer and 2.5 heads). Figure 2 shows a mucosal carcinoma with U of Taq DNA polymerase. The human Ets-1 PCR negative staining for Ets-1. 1934 Nakayama et al 4/P December 1996, Vol. 149, No. 6

Table 1. Histological Types of Gastric Adenocarcinomas and Immunobistochbenical Ets-I Staining (110 Cases) n ++ Total carcinomas 110 40 (36.4%) 23 (20.9%) 47 (42.7%) Papillary adenocarcinoma 13 7 (53.8%) 3 (23.1%) 3 (23.1%) Tubular adenocarcinoma Well differentiated 26 14 (53.8%) 2 (7.7%) 10 (38.5%) Moderately differentiated 27 4 (14.8%) 7 (25.9%) 16 (59.3%) Poorly differentiated adenocarcinoma Solid type 14 3 (21.4%) 5 (35.7%) 6 (42.9%) Nonsolid type 13 1 (7.7%) 2 (15.4%) 10 (76.9%) Signet-ring cell carcinoma 10 8 (80.0%) 2 (20.0%) 0 (0.0%) Mucinous adenocarcinoma 7 3 (42.8%) 2 (28.6%) 2 (28.6%) Adenoma 1 1 11 (100.0%) 0 (0.0%) 0 (0.0%) Normal mucosa 15 15 (100.0%) 0 (0.0%) 0 (0.0%) *See Materials and Methods for classification of staining intensity.

Figure 1. A: Hematoxylin and eosin staining of invasive adenocarcinoma in the human stomach. B: Immunohistochemistry studies of Ets-1 reveals positive staining in cytoplasm and nucleus in carcinoma cells. C: Ets-1 protein is located dominantly in the nucleus. In this case, Ets-1 is partially expressed both in the cytoplasm and nucleus (arrowheads). Immunoalkalinephosphatase staining; Mag- nification, X 100 (A and B) and x200 (C). Ets-1 Expression in Gastric Carcinoma 1935 AJP December 1996, Vol. 149, No. 6

...... - .. ,. ::.

iI': ;:,. .:"' :*

M*_

.*.,> :: '

,w,.4.

* :.: *:. .# i ...}v .! 7 '-** tF'-

.. ' .' *: °E;DS' ::.ko ' a , g s * &L #. wr i,_ild7 A....t, :* -;ff ^ -< J .R.

' | '..:4 sg.

b $.

rirq'> *S.::. :: * .i 1:+ w -.@ :.p;, :: ;. Figure 2. A: Hematoxylin and eosin staining of mucosal adenocarcinoma in the human stomach. Arrowheads show the muscularis mucosa. B: Immunohistochemistry studies ofEts-1. Carcinoma cells- are negative for Ets-1. Immunoalkaline phosphatase staining; Magnification, X ..100.jjE..,c.

The degree of immunoreactivity appears to be ences between mucosal carcinomas and the other correlated with the degree of tumor infiltration (Table carcinomas (P < 0.001; Table 2). In the invasive 2). Staining for ETS-1 was mainly negative for tumors carcinomas, the staining intensity in signet-ringiZZcell with mucosal infiltration (only 3 of 26 were positive, carcinomas was statistically lower than with the other 11.5%), and there was no case of strong staining (0 types of carcinoma (P < 0.05). Also, the invasive of 26), whereas 71.9% of submucosal infiltrative tu- front and/or the peripheral parts of the primary tumor mors were strongly Ets-1 immunoreactive (23 of 32). were intensely stained compared with the superficial In tumors infiltrating the proprial muscle layers, the and central parts of the tumor in almost all cases. subserosa, and the serosal surface, Ets-1 expres- Ets-1 staining intensity was compared in the pri- sions were frequently strongly positive (44 of 52, mary and metastatic tumors (Table 3). Ets-1 expres- 84.6%). Statistical analysis showed significant differ- sion in primary carcinomas correlated statistically

Table 2. Invasive Grades of Gastric Carcinomas and Ets-1 Immunohistochemistry (110 Cases) n + ++ P value m 26 23 (88.5%) 3 (11.5%) 0 (0.0%) <0.001 sm 32 9 (28.1%) 9 (28.1%) 14 (43.8%) mp 14 2 (14.3%) 3 (21.4%) 9 (64.3%) ss 16 3 (18.8%) 3 (18.8%) 10 (62.5%) se 22 3 (13.6%) 5 (22.7%) 14 (63.6%) Total 110 40 (36.4%) 23 (20.9%) 47 (42.7%) m, mucosal carcinomas; sm, submucosal infiltrative carcinomas; mp, carcinomas invading the proprial muscle layers; ss, carcinomas reaching the subserosa; se, carcinomas penetrating the serosal surface. 1936 Nakayama et al AJP December 1996, Vol. 149, No. 6

Table 3. Expression qf Ets-1 in Primary Adenocarcinomas with or without Lymph Node Metastasis (97 Cases) + P value Lymph node metastasis Present 41 7 (17.1%) 10 (24.4%) 24 (58.5%) <0.001 Absent 56 30 (53.6%) 10 (17.9%) 16 (28.6%) with the presence of lymph node metastasis (P < ulcer progression. In advanced gastric carcinoma, 0.001). the 5-year survival rate is approximately 20% in the As with the antigenic intensity shown by immuno- United States and 45% in Japan when a potentially staining, Ets-1 mRNA expression demonstrated by in curative operation has been performed.2122 How- situ hybridization was positive in both the cytoplasm ever, early gastric cancer has a relatively higher and the nucleus of gastric adenocarcinomas (Figure 5-year survival rate. The 5-year survival rates after a 3B). No specific hybridization was observed with the potentially curative operation for mucosal and sub- sense-labeled probe. RNAse treatment of the sec- mucosal cancer are almost 100 and 92%, respec- tions hybridized with the Ets-1 oligonucleotide probe tively, in Japan.22 This difference in prognosis is due yielded no positive signals. to the presence of lymphatic infiltration and metas- The results from RT-PCR of Ets-1 mRNA in the tasis. Lymphatic infiltration was present in 1.3% of normal gastric mucosa and in human gastric carci- mucosal carcinomas and in 67.9% of submucosal noma cell lines are shown in Figure 4. In comparison carcinomas.1 Lymph node metastasis occurred in with normal gastric mucosal cells, which did not 1.8% of mucosal carcinomas and in 12.7% of sub- express Ets-1 mRNA, all gastric carcinoma cell lines mucosal carcinomas.1 Therefore, for purposes of expressed Ets-1 mRNA. 3-actin mRNA, a control to prognosis, it is important to discriminate whether the demonstrate the equivalent amounts of tissue RNA, carcinoma cells have invaded beyond the muscu- was used for cDNA synthesis and was detected in all laris mucosa. In this study, the overexpression of samples. Ets-1 promoted the invasion of mucosal carcinoma into the submucosa, and the degree of expression Discussion was correlated to the extent of gastric carcinoma invasion. In gastric carcinoma, the invasive grade is one of the There is expression of the Ets family of genes most significant factors determining a patient's prog- during embryonic life, particularly in the stomach, nosis. The muscularis mucosa plays an important and this expression is dramatically reduced in later role in defending against carcinoma invasion and fetal age.3'4 In our study, Ets-1 was not expressed in

Figure 3. A: Hematoxylin and eosin staining of invasive adenocarcinoma in the hbumani stomach. B: Using the in SituL hbhridizationi method, Ets-I nmRNVA is expressed in citoplasm of carcinoma cells. Magnification, X200. Ets-1 Expression in Gastric Carcinoma 1937 AJP December 1996, Vol. 149, No. 6

stromelysin,28 parathyroid hormone-related protein,29 urokinase-type plasminogen activator, and collagenase-1 .6,13 The expression of these sub- A -- 274 bp. stances has also been observed in gastric carcinoma cells,30-33 and they may play important roles in tumor invasion and progression. It was necessary to determine whether Ets-1 is produced by mesenchymal cells or carcinoma cells. Ets-1 is thought to be involved not only in tumor invasion but in connective tissue remodel- B - 313 bp. ing.34 We observed increased Ets-1 expression during vascular smooth muscle cell migration and/or proliferation induced by serum stimulation in vitro and by balloon injury in vivo.17 The Ets-1 M 1 2 3 4 5 6 mRNA is known to be expressed in stromal fibro- Figure 4. RT-PCR analysis ofEts-1 mRNA expression in human gastric cells.35'36 The expres- cells using the specific primerpairs predicted to amnplifyfragment size blasts around carcinoma on the right. A: Ets-1. B: /3-actin as internal control. Total RNA was sion of the Ets-1 gene has also been demonstrated preparedfromfourgastric carcinoma cell lines (lanes 1 to 4) and two In this normal gastric mucosal tissue specimens (lanes 5 and 6). Size marker in vascular sarcomas37 and astrocytomas.38 (lane M) is pBR322/MspI digest. study, however, we detected Ets-1 mRNA in the gastric carcinoma cells themselves but not in- the normal gastric epithelium or in gastric adeno- tensely in stromal fibroblasts, as shown by immu- mas. In mucosal adenocarcinomas, its expression nohistochemistry and in situ hybridization. RT-PCR was weak but was enhanced in submucosal carci- of the Ets-1 mRNA from the four human carcinoma nomas and in the more invasive carcinomas. This cell lines confirmed that it is produced in the car- suggests that Ets-1 plays a role in local invasion to cinoma cells themselves. These findings suggest the submucosa in gastric carcinomas and in the that Ets-1 is produced by carcinoma cells and may development of invasive characteristics as the last play a critical role in the penetration of gastric step in carcinogenesis. carcinoma cells. Although Ets-1 expression was generally en- In conclusion, our results suggest that Ets-1 ex- hanced in the more advanced cases, its expression pression may be one of the factors triggering the was not increased in approximately 10% of the inva- invasion of the submucosa by gastric carcinomas. sive gastric carcinomas. Five of eight of these neg- The overexpression of the Ets-1 proto-oncogene ative cases were signet-ring cell carcinomas product may be the last step in the multi-step carci- (62.5%). Staining intensity in signet-ring cell carcinogenesis of human gastric carcinomas. noma was statistically lower than that in the other types of advanced carcinoma (P < 0.05). In this respect, we suggest that signet-ring cell carcinomas have different invasive mechanisms than other types References of carcinoma. 1. Goseki N, Koike M, Yoshida M: Histopathologic char- The product of Ets-1 is localized in the nucleus acteristics of early stage esophageal carcinoma: a and binds to DNA.23 However, in other reports, the comparative study with gastric carcinoma. Cancer Ets-1 protein was detected in the cytoplasm24 or 1992, 69:1088-1093 both in the cytoplasm and in the nucleus in human 2. Leprince D, Gegonne A, Coll J, de Taisne C, Schnee- colorectal carcinoma cell lines.25 In this study, we berger A, Lagrou C, Stehelin D: A putative second showed immunohistochemically that Ets-1 protein cell-derived oncogene of avian leukemia retrovirus was widely expressed in the cytoplasm of carci- E26. Nature 1983, 306:395-397 noma cells as well as in the nucleus. This is the first 3. Kola I, Brookes S, Green AR, Garber R, Tymms M, report and analysis of Ets-1 production in the gas- Papas T, Seth A: The Etsl transcription factor is widely and is tric carcinoma cell itself. We believe that Ets-1 expressed during murine embryo development associated with mesodermal cells involved in morpho- protein is overproduced in the cytoplasm and genetic processes such as organ formation. Proc Natl binds to DNA in the nucleus of gastric carcinoma Acad Sci USA 1993, 90:7588-7592 cells. Recently, it was shown that Ets-1 protein 4. Vandenbunder B, Pardanaud L, Jaffredo T, Mirabel regulates the gene expression of some cytokines MA, Stehelin D: Complementary patterns of expression and peptides such as Fos and Jun,26 integrin,27 of c-etsl, c-myb, and c-myc in the blood-forming sys- 1938 Nakayama et al AJP December 1996, Vol. 149, No. 6

tem of the chick embryo. Development 1989, 106:265- Targeted cytogenetic analysis of gastric tumors by in 274 situ hybridization with a set of chromosome specific 5. Lewin B: Oncogenic conversation by regulatory DNA probes. Cancer 1990, 66:491-497 changes in transcription factors. Cell 1991, 64:303-312 21. Morson BC, Dawson IMP: Morson, Dawson's Gastroin- 6. Vandenbunder B, Wernert N, Queva C, Desbiens X, testinal Pathology, ed 3. Edited by BC Morson, IMP Stehelin D: Does the transcription factor c-etsl take Dawson. London, Blackwell, 1990, p 159 part in the regulation of angiogenesis and tumor inva- 22. Kodama Y, Sugimachi K, Soejima K, Matsusaka T, sion? Folia Biol (Prague) 1994, 40:301-313 Inokuchi K: Evaluation of extensive lymph node dissec- 7. Liotta LA, Stetler-Stevenson WG: Metalloproteinases tion for carcinoma of the stomach. World J Surg 1981, and cancer invasion. Semin Cancer Biol 1990, 1:99- 5:241-248 106 23. Pognonec P, Boulukos KE, Ghysdael J: The c-ets-1 8. Matrisian LM, Bowden TG: Stromelysin/transin tumor protein is chromatin associated and binds to DNA in progression. Semin Cancer Biol 1990, 1:107-115 vitro. Oncogene 1989, 4:691-697 9. Sloane BF: Cathepsin B cystatins: evidence for a role in 24. Fujiwara S, Fisher RJ, Seth A, Bhat NK, Showalter SD, cancer progression. Semin Cancer Biol 1990, 1:137- Zweig M, Papas TS: Characterization and localization 152 of the products of the human homologs of the v-ets 10. Rochefort H: Biological and clinical significance of ca- oncogene. Oncogene 1988, 2:99-103 thepsin D in breast cancer. Semin Cancer Biol 1990, 25. Koizumi S, Fisher RJ, Fujiwara S, Jorcyk C, Bhat NK, 1:153-160 Seth A, Papas TS: Isoforms of the human Ets-1 protein: 11. Kane SE, Gottesman MM: The role of cathepsin L in generation by alternative splicing and differential phos- malignant transformation. Semin Cancer Biol 1990, phorylation. Oncogene 1990, 5:675-681 1:127-136 26. Wasylyk B, Wasylyk C, Flores P, Begue A, Leprince D, 12. Blasi F, Verde P: Urokinase-dependent cell surface Stehelin D: The c-ets proto-oncogenes encode tran- proteolysis and cancer. Semin Cancer Biol 1990, scription factors that cooperate with c-Fos and c-Jun 1:117-1 26 for transcriptional activation. Nature 1990, 346:191- 13. Nerlov C, De Cesare D, Pergola F, Caraccilo A, Blasi F, 193 Johnsen M, Verde P: A regulatory element that medi- 27. Rosen GD, Barks JL, lademarco MF, Fisher RJ, Dean ates co-operation between a PEA3-AP-1 element and DC: An intricate arrangement of binding sites for the an AP-1 site is required for phorbol ester induction of Ets family of transcription factors regulates activity of urokinase enhancer activity in HepG2 hepatoma cells. the a-4 integrin gene promoter. J Biol Chem 1994, EMBO J 1992, 11:4573-4582 269:15652-15660 14. Japanese Research Society for Gastric Cancer: Japa- 28. Higashino F, Yoshida K, Noumi T, Seiki M, Fujinaga K: nese Classification of Gastric Carcinoma (First English Ets-related protein E1A-F can activate three different Edition). Edited by Japanese Research Society for matrix metalloproteinase gene promoters. Oncogene Gastric Cancer. Tokyo, Kanehara Press, 1995 1995, 10:1461-1463 15. Takeshima E, Hamaguchi M, Watanabe T, Akiyama S, 29. Dittmer J, Gegonne A, Giltin SD, Ghysdael J, Brady JN: Kataoka M, Ohnishi Y, Xiao H, Nagai Y, Takagi H: Regulation of parathyroid hormone-related protein Aberrant elevation of tyrosine-specific phosphorylation (PTHrP) gene expression: Spl binds through an in- in human gastric cancer cells. Jpn J Cancer Res 1991, verted CACCC motif and regulates promoter activity in 82:1428-1435 cooperation with Etsl. J Biol Chem 1994, 269:21428- 16. Koshikawa N, Yasumitsu H, Umeda M, Miyazaki K: 21434 Multiple secretion of matrix serine proteinases by hu- 30. Nomura N, Yamamoto T, Toyoshima K, Ohami H, Aki- man gastric carcinoma cell lines. Cancer Res 1992, maru K, Sasaki S, Nakagami Y, Kanauchi H, Shoji T, 52:5046-5053 Hiraoka Y, Matsui M, Ishizaki R: DNA amplification of 17. Hultgardh-Nilsson A, Cercek B, Wang J-W, Naito S, the c-myc and c-erbB-1 genes in a human stomach Lovdahl C, Sharifi B, Forrester JS, Fagin JA: Regulated cancer. Jpn J Cancer Res 1986, 77:1188-1192 expression of the Ets-1 transcription factor in vascular 31. Fujita S, Suzuki H, Kinoshita M, Hirohashi S: Inhibition smooth muscle cells in vivo and in vitro. Circ Res 1996, of cell attachment, invasion, and metastasis of human 78:589-595 carcinoma cells by anti-integrin f31 subunit antibody. 18. Dennis K, McWilliams MJ, Lapis P, Lautenberger A, Jpn J Cancer Res 1992, 83:1317-1326 Schweinfest CW, Papas TS: Mammalian ets-1 and ets-2 32. McDonnall S, Navre M, Coffey RJ Jr, Matrisian LM: genes encode highly conserved proteins. Proc Natl Expression and localization of the matrix metallopro- Acad Sci USA 1988, 85:7862-7866 teinase pump-1 (MMP-7) in human gastric and colon 19. Ohtsuru A, Nagataki S, Koji T, Tamaoki T: Analysis of carcinomas. Mol Carcinogen 1991, 4:527-533 a-fetoprotein gene expression in hepatocellular carci- 33. Abdeen 0, Pandol SJ, Burton DW, Deftos LJ: Parathy- noma and liver cirrhosis by in situ hybridization. Cancer roid hormone-related protein expression in human gas- 1988, 62:1105-1112 tric adenocarcinomas not associated with hypercalce- 20. Van Dekken H, Pizollo JG, Kelsen DP, Melamed MR: mia. Am J Gastroenterol 1995, 90:1864-1867 Ets-1 Expression in Gastric Carcinoma 1939 AJP December 1996, Vol. 149, No. 6

34. Liotta LA, Steeg PS, Stetler SWG: Cancer metastasis plasminogen activator genes in lung carcinomas. Am J and angiogenesis: an imbalance of positive and neg- Pathol 1995, 147:1298-1310 ative regulation. Cell 1991, 64:327-336 37. Wernert N, Raes MB, Lassalle P, Dehouck MP, Gosse- 35. Wernert N, Gilles F, Fafeur V, Bouali F, Raes M-B, Pyke lin B, Vandenbunder B, Stehelin D: c-etsl proto-onco- C, Dupressoir T, Seitz G, Vandenbunder B, Stehelin D: gene is a transcription factor expressed in endothelial cells Stromal expression of c-Etsl transcription factor corre- during tumor vascularization and other forms of angiogenesis in humans. Am J Pathol 1992, 140:119- lates with tumor invasion. Cancer Res 1994, 54:5683- 127 5688 38. Amouyel P, Gegonne A, Delacourte A, Defossez A, 36. Bolon I, Gouyer V, Devouassoux M, Vandenbunder B, Stehelin D: Expression of ETS proto-oncogene in as- Wernert N, Moro D, Brambilla C, Brambilla E: Expres- trocytes in human cortex. Brain Res 1988, 447:149- sion of c-ets-1, collagenase 1, and urokinase-type 153