Tyrosine kinases and gastric cancer - Nature

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Oncogene (2000) 19, 5680 ± 5689 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc

Tyrosine kinases and gastric cancer Wen-chang Lin*,1,5, Hsiao-Wei Kao1, Daniel Robinson2, Hsing-Jien Kung2, Chew-Wun Wu3 and Hua-Chien Chen*,4,5 1

Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, Republic of China; 2Department of Biological Chemistry, UC Davis School of Medicine, UC Davis Cancer Center, Sacramento, California, CA 95817, USA; 3Department of Surgery, Veterans General Hospital-Taipei and School of Medicine, National Yang-Ming University, Taipei 112, Taiwan, Republic of China; 4Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Taipei 115, Taiwan, Republic of China

Carcinoma of the stomach is one of the most prevalent cancer types in the world today. Two major forms of gastric cancer are distinguished according to their morphological and clinicopathological classi®cations (well dierentiated/intestinal type and poorly dierentiated/diuse type), characteristics that could also be attributed to the altered expression of dierent types of oncogenes or tumor suppressor genes. Signi®cant dierences exist for gastric cancer incidence comparing people of dierent ethnic origins, implicating various genetic and epigenetic factors for gastric oncogenesis. There are only a limited number of molecular markers available for gastric cancer detection and prognostic evaluation, among which are tyrosine kinases. There is convincing evidence that tyrosine kinases are involved in oncogenesis and disease progression for many human cancers. Ampli®cations of certain tyrosine kinases (cmet, k-sam and erbB2/neu) have been associated with human gastric cancer progression. Alternatively spliced transcripts and enhanced protein-expression levels for some of these tyrosine kinases are correlated with clinical outcomes for gastric cancer patients. With advent of high throughput techniques, it is now possible to detect nearly all expressed tyrosine kinases in a single screen. This increases the chance to identify additional tyrosine kinases as predictive markers for gastric cancers. In this article, we will ®rst review the literature data concerning certain tyrosine kinases implicated in gastric carcinogenesis and then summarize more recent work which provide comprehensive tyrosine kinase pro®les for gastric cancer specimens and cell lines. Two new gastric cancer molecular markers (tie-1 and mkk4) have been identi®ed through the use of these pro®les and demonstrated eective as clinical prognostic indicators. Oncogene (2000) 19, 5680 ± 5689. Keywords: gastric cancer; protein-tyrosine kinase pro®le Introduction Gastric cancer remains one of the most common cancer types in the world today, despite the fact that its incidence has gradually declined in many countries (Neugut et al., 1996). Gastric cancer is more prevalent in the Andean region of South America and in the Far *Correspondence: W-c Lin and H-C Chen 5 W-c Lin and H-C Chen share the corresponding authorship

East (including Taiwan) and it is still the leading cause of cancer death for several countries (e.g. Japan, Chile) (Parkin et al., 1988). In Taiwan, gastric cancer ranks as the fourth-highest cause of cancer-related death, with a mortality rate of 10.72 per 100 000 for the year 1999 (Department of Health, 2000). In the United States, a country with relatively low gastric cancer incidence, the mortality rate for 1997 was 3.4 and 7.5 per 100 000 for Caucasians and African Americans, respectively (Ries et al., 2000). Therefore, given this regional variation, it seems reasonable to suggest that genetic variations/ factors contribute to gastric cancer oncogenesis for dierent ethnicities. Although diet is an important factor in the development of gastric and colorectal cancers, the dierence of colorectal cancer incidence for Caucasians and African Americans is not as signi®cant as that of gastric cancer. Again, this suggests that genetic factors may play a signi®cant role in the development of gastric cancers, together with well-known epigenetic factors, such as diet and life style. Another intriguing observation is that female gastric cancer incidence is as low as half that for males (Ries et al., 2000) ± noted both for Taiwan (43%), a high incidence region, and the United States (44%), a low incidence region. The clinical outcome for gastric cancer patients remains poor, with a 5-year survival rate of only about 20% (all stages) (Ries et al., 2000), and a mere 40% of all patients responding to surgical intervention (Ajani et al., 1995). Surgical resection is the only eective curative modality for primary treatment of gastric cancer, with the overall cumulative 5-year survival rate improving to 62.4% for 362 patients in one report (Wu et al., 2000b). Most gastric cancer patients are diagnosed with advanced diseases, however, resulting in a dismal prognosis and underscoring the importance of the identi®cation of useful diagnostic and prognostic markers for gastric cancers. For this report, we summarize data contributing to the identi®cation of tyrosine kinases as potential markers for gastric cancers. To provide background information, we also brie¯y summarize the current knowledge for reported genetic alterations of gastric cancers. Known genetic alterations for gastric cancer With the aid of continuing advances in molecular biology, recent research eorts have identi®ed a number of genetic alterations associated with gastric cancer oncogenesis. These genetic changes include

Tyrosine kinases in human gastric cancer W-c Lin et al

genetic instability represented by microsatellite instability (CA repeats), reactivation of telomerase activity, inactivation of tumor-suppressor genes (p53, APC, DCC, MCC), and activation of oncogenes (VEGF, cyclin E, K-ras, erbB2, K-sam, c-met, TGF-bRII). Tahara has summarized and discussed these genetic alterations in great detail in his review articles (Tahara, 1995a,b; Yasui et al., 1999, 2000). Although some genetic changes are common (microsatellite instability, telomerase activity, CD44 splicing variants, and p53 mutation status), it is interesting to note that several dierences for genetic alterations have been observed comparing the poorly dierentiated (mostly diuse-type carcinoma) and well-dierentiated (mostly intestinal-type carcinoma) gastric cancers (Ming, 1998). In well-dierentiated gastric cancers, inactivation of APC and DCC tumor-suppressor genes by LOH (loss of heterozygocity) and mutation is prevalent, as well as aberrant expression patterns of nm23 and bcl-2 genes. A previous investigation of eight commonly studied tumor markers (EGF, EGFR, TGFa, p53, erbB2, cripto, NM23), comparing British and Japanese gastric cancer samples, revealed that only two markers generated dierential results. Immunoreactivity was reportedly higher for erbB2 in British gastric cancer specimens, while NM23 immunoreactivity was positive for the greater portion of Japanese samples (Livingstone et al., 1995). Since these markers are common components in cellular growth and signal pathways, it is perhaps not surprising that similar expression pro®les have been revealed for gastric cancers comparing two dierent ethnicities. In other studies, there is no dierence in erbB2 expression among British or Japan gastric cancers (McCulloch et al., 1995, 1997). Loss of E-cadherin and catenin genes is predominant, however, for the poorly dierentiated gastric cancers (Ming, 1998). This evidence strongly supports the view that distinct genetic pathways are involved in the development of the two major types of gastric cancers. Regarding oncogene activation for gastric cancer carcinogenesis, K-ras mutation has been determined for the well-dierentiated gastric types, however, with a much lower frequency than for colorectal cancers (Ming, 1998). Tyrosine kinases and gastric cancer Tyrosine-kinase receptors are key molecules in signaling pathways leading to growth and dierentiation of normal cells (Marshall, 1995). Aberrant expression of tyrosine kinases as re¯ected by the aberrant tyrosine phosphorylation in gastric cancer cells was reported (Takeshima et al., 1991). Three receptor-tyrosine kinases, c-met, k-sam, and erbB2/neu, which are frequently implicated in epithelial cancers were most extensively studied in gastric carcinomas. While c-met (HGF receptor) is over-expressed/ampli®ed for advanced gastric cancers, for both well-dierentiated and poorly dierentiated gastric carcinomas, k-sam (FGF receptor 2) activation is more speci®c for the poorly dierentiated variants. By contrast, ampli®cation of the erbB2/neu gene is often revealed for well-dierentiated gastric carcinomas (Ming, 1998). Thus, subversion of dierent signal pathways may contribute to the progression of dierent types of gastric cancers.

This review consists of two parts. In the ®rst, the involvement of the c-met, k-sam and erbB-2 in gastric cancers as reported in the literature will be reviewed. The discussion includes the characterization of these genes in gastric cancers, in terms of gene ampli®cation at the DNA level, alternatively spliced transcripts, protein expression as studied by immunohistochemistry, and biological activities of these receptor tyrosine kinases. In the second part, the discussion will be extended to the entire collection of tyrosine kinases expressed in gastric cancers, i.e., the tyrosine kinase pro®les of gastric cancers. The cited literature is not meant to be inclusive, but rather, representative.

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HGF receptor tyrosine kinase DNA amplification The c-met receptor tyrosine kinase was ®rst isolated from an osteogenic sarcoma cell line (Cooper et al., 1984), and subsequently identi®ed as the receptor for hepatocyte growth factor (HGF) (see, Furge et al. this issue). The c-met functional unit consists of an extracellular alpha subunit and a transmembrane beta subunit. Ampli®cation of the c-met gene has been demonstrated for gastric cancer tissues (Kuniyasu et al., 1992), especially for the scirrhous type. Coampli®cation of c-met and c-myc oncogenes have also been reported for gastric cancers (Seruca et al., 1995). Alternatively spliced transcripts Rearrangement fusion of the tpr-met gene is one of the activation mechanisms reported to produce aberrant transcripts (Park et al., 1986). In addition, isoforms of the c-met transcripts, generated by alternative splicing, have been observed (Rodrigues et al., 1991), and a special 6.0 Kb transcript, demonstrated for gastric cancer cells, has been correlated with cancer progression (Kuniyasu et al., 1993). The biological activities for dierent isoforms, especially alterations in the extracellular ligand-binding region, have not been elucidated as yet. In KATO III gastric cancer cells, alternatively spliced transcripts, generating in-frame deletion for the c-met related receptor tyrosine kinase (ron), have also been demonstrated for the extracellular domain (Collesi et al., 1996; Okino et al., 1999). It is likely that these alterations may alter ligand-binding anity, and are subsequently involved in oncogenic activation. Protein localization by immunohistochemistry It has been demonstrated that the numbers of functional c-met receptors are more important than overall numbers of receptors for the growth of gastric cancer cells (Liu et al., 1997). Nevertheless, a high positive rate (40 ± 80%) for c-met immunoreactivity has been observed in several reports (Park et al., 2000; Taniguchi et al., 1998; Wu et al., 1998). Simultaneous detection of HGF expression and c-met immunoreactivity has also been noted for the majority of gastric cancer specimens (Park et al., 2000; Wu et al., 1998), indicating a possible autocrine or paracrine mechanism. This proposition is supported by growth inhibition for gastric cancer cell lines with anti-sense oligonucleotides Oncogene


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blocking c-met gene expression (Kaji et al., 1996). For gastric cancer patients, co-expression of c-met and erbB2 proteins have been associated with poorer survival rates compared with over-expression of either one (Nakajima et al., 1999). Co-ampli®cation of c-met, k-sam or erbB2 genes at the genomic level, however, has not been observed for most of the gastric cancer specimens examined (Hara et al., 1998; Tsugawa et al., 1998; Tsujimoto et al., 1997). This indicates that activations of c-met, k-sam and erbB2 are likely to be independently regulated. As stated previously, ampli®cation of these receptor tyrosine kinases was observed separately for well-dierentiated (k-sam) and poorly dierentiated (erbB2) gastric cancers (Ming, 1998).

FGF receptor tyrosine kinase DNA amplification The k-sam receptor tyrosine kinase was ®rst identi®ed from the KATO III gastric cancer cell line, and determined as the type 2 receptor for the ®broblast growth factor (FGF) or keratinocyte growth factor (KGF) (Hattori et al., 1990). Ampli®cation of the k-sam gene has been observed mainly for poorly dierentiated gastric cancer tissues (Ming, 1998), correlating with poor prognosis (Hattori et al., 1996). Alternatively spliced transcripts At least four major alternatively spliced k-sam transcripts (k-sam I ± IV) have been previously observed (Katoh et al., 1992). Alterations in the extracellular ligand binding region for k-sam I and ksam II transcripts confer ligand speci®city between basic FGF and KGF (Ishii et al., 1994; Miki et al., 1992). The coding sequences of K-sam III and k-sam IV transcripts lack the transmembrane domain and therefore encode for soluble forms of the FGF receptor, with k-sam III transcript still retaining the sequence encoding the kinase domain (Katoh et al., 1992). Soluble k-sam protein has indeed been detected in the culture medium of KATO III cells (Kishi et al., 1994). For KATO III gastric cancer cells, a major transcript (k-sam IIC3) encoding a truncated carboxylterminus FGFR 2 protein has been demonstrated to be transforming (Itoh et al., 1994). This protein is generated by an alternative splicing mechanism and possibly loses a regulatory tyrosine residue at the Y769 position due to truncation, similar to src oncogene activation. Recently, several additional novel k-sam transcripts, derived from alternative-splicing (Ueda et al., 1999a) or short homology-mediated recombination (Ueda et al., 1999b) have been discovered for scirrhous-type gastric cancer cell lines. Such truncated forms of the k-sam receptor alter the induceddierentiation pathways for transfected cells (Ishii et al., 1995), indicating that alternatively spliced k-sam products may have physiologically distinct functions and be involved in the regulation of cellular dierentiation. It has also been demonstrated that FGFR 4, another member of the FGFR tyrosine kinases, expressed Oncogene

alternatively spliced transcripts that generate a soluble form of FGFR 4 for human gastric cancer cells (Takaishi et al., 2000). The biological signi®cance for these soluble forms of receptor tyrosine kinases still needs further elucidation. It is possible that these soluble forms regulate membrane-type receptor functions by competing with ligand-binding activity, or may serve as a ligand reservoir as seen for cytokine receptors (Jung et al., 1999; Saily et al., 1999; Sayani et al., 2000; Scott and Weiss, 2000). Protein localization by immunohistochemistry As articulated through immunohistochemistry, k-sam protein is detected in about 50% of poorly dierentiated gastric cancers, but not in well-dierentiated gastric cancers (Hattori et al., 1996). It has been determined that k-sam protein expression is associated with the malignant progression of gastric cancer, however, no k-sam autocrine/paracrine mechanism for gastric cancers has been observed. The proteinexpression pattern for basic FGF and bek/k-sam does not correlate with tumor stages of gastric cancers (Takahashi et al., 1996). Since the FGFs and their receptors belong to large gene families (Gospodarowicz, 1989; Jaye et al., 1992), detailed expression pro®les for all FGFs and FGFRs are needed to achieve a full appreciation about their involvement in gastric cancers.

The EGF/ErbB receptor family of tyrosine kinases DNA amplification Among receptor tyrosine kinases, the EGF receptor gene family including EGFR and erbB2, is the most frequently implicated in human cancers. Ampli®cation of EGFR and erbB2 genes for human gastric cancers has been determined at around 3*5% and 10*20%, respectively (Albino et al., 1995; Hirono et al., 1995; Sato et al., 1997; Tahara, 1995a; Tsugawa et al., 1993; Yoshida et al., 1989; Hung and Lau, 1999). All erbB2ampli®ed gastric cancer cells reveal enhanced erbB2 protein expression patterns (Ooi et al., 1998). Coampli®cation of gastrin and erbB2 has been reported for intestinal-type gastric cancers (Vidgren et al., 1999), probably because they are located close together at chromosome 17q. Alternatively spliced transcripts In situ hybridization detection of the EGFR message revealed that its high expression is associated with a higher frequency of gastric-cancer recurrence (Anzai et al., 1998). Activated erbB family receptor tyrosine kinases as well as the soluble form of the receptor proteins, can be generated by alternatively spliced messages for all erbB family members (EGFR, erbB2, erbB3, and erbB4) (Flickinger et al., 1992; Gebhardt et al., 1998; Ilekis et al., 1995; Katoh et al., 1993; Kwong and Hung, 1998; Lee and Maihle, 1998; Sawyer et al., 1998; Scott et al., 1993). Secreted erbB2 and erbB3 splicing variants have been identi®ed for human gastric cancer cells (Katoh et al., 1993; Scott et al., 1993). Elevated level of soluble EGFR and erbB2 proteins,


possibly resulting from alternatively spliced transcripts or proteolytic cleavage, are detectable in the serum of gastric cancer patients (Choi et al., 1997; Kono et al., 2000; Perng et al., 1994; Molina et al., 1997). As discussed above, soluble tyrosine kinase receptors may play a regulatory role for receptor signaling. On the other hand, the elevated level of soluble receptors could be a result of shedding from tumor cells overexpressing these receptors. In either case, they may be pro®tably used as markers for gastric cancer cells. Protein localization by immunohistochemistry Immunoreactivity to EGFR antibodies is demonstrated for 30 ± 50% of human gastric cancers (Jonjic et al., 1997; Yonemura et al., 1989) and correlated with poor prognosis, however, EGFR immunoreactivity is not an independent prognostic factor according to multivariate analysis (Santoro et al., 1997). For erbB2 protein, positive rates for erbB2 immunoreactivity range from 10 to 50% for the human gastric cancers that have been studied, and is higher for welldierentiated gastric cancers (Kameda et al., 1990; Lee et al., 1996; Motojima et al., 1994; Orita et al., 1997; Uchino et al., 1993; Yonemura et al., 1991a). The expression of erbB2 protein is a signi®cant independent risk factor for long term survival, disease recurrence and lymph-node metastasis (Lin et al., 1995; Motojima et al., 1994; Orita et al., 1997; Sanz-Ortega et al., 2000; Yonemura et al., 1991a). Since homo-dimerization or hetero-dimerization between members of the EGFR family is required for activation, the presence of other erbB family members in the same cell can profoundly in¯uence the outcome of the signals. The expression pattern of erbB family members (EGFR, erbB2, erbB3 and erbB4) has been examined in several studies. Co-expression of EGFR, erbB2 and erbB3 all three receptors has been demonstrated in a signi®cant portion of gastric cancers but not in chronic gastritis (Slesak et al., 1998). Expression of ErbB4 is less common and has been noted for gastric cancers in one study (Kataoka et al., 1998). Autocrine and paracrine pathways have been demonstrated for the erbB family. The presence of the EGFR ligands (EGF and TGF-a) and the erbB2/ erbB3 ligand (heregulin-a), with their respective receptors, have been reported for the human gastric mucosa (Beauchamp et al., 1989) and gastric cancer tissues (Tokunaga et al., 1995). Heregulin-a is expressed in ®broblasts associated with stomach tissue, but is capable of inducing proliferation of gastric cancer cells (Noguchi et al., 1999). Proliferation index, tumor invasion and metastasis frequency are higher for gastric cancer patients expressing both EGFR and EGF/TGF-a in cancer tissues (Borlinghaus et al., 1993; Filipe et al., 1995; Karameris et al., 1993; Yasui et al., 1988; Yonemura et al., 1991b, 1992; Yoshida et al., 1990). Although both EGF binding activity and EGFR immunoreactivity are important parameters for human gastric cancer progression, there is no signi®cant correlation between these two elements. Likewise, the expression level of functionally activated receptors should be a more reliable biomarker than the total numbers of receptors (Turner et al., 2000) and higher EGFR kinase activity has indeed been observed for gastric cancer tissues compared to adjacent mucosa

(Kameda et al., 1992; Masaki et al., 2000). Higher EGF-binding activity also correlates with potential of growth for gastric tumor cells in nude mice (Kiyama et al., 1992). This indicates that activation of expressed EGFR family receptors is one of the critical elements in the growth of gastric cancers. Blocking EGFR expression with antisense vectors inhibits the growth of gastric cancer cells (Hirao et al., 1999). Antibodies against erbB2/neu have been demonstrated eective for reducing the tumor burden and prolonging survival of treated animals with gastric cancers (Karameris et al., 1993; Ohnishi et al., 1995; Tokuda et al., 1996). Targeting erbB2-expressing cells with conjugated immunoliposomes containing chemotherapeutic reagents has also proven useful (Suzuki et al., 1995). Recently, the recombinant humanized anti-HER2 antibody, herceptin, has been adopted for clinical use for treatment of several human cancers (Agus et al., 1999; Baselga et al., 1998; Stebbing et al., 2000). Its ecacy in gastric cancers has yet to be determined.

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Other protein tyrosine kinases in gastric cancer There are several other protein tyrosine kinases that have, in a limited fashion, been previously examined for human gastric cancers. These include src, vascular endothelial growth factor receptor (KDR), hek5 (erk), abl, nerve growth factor receptor (trkA), and stem cell factor receptor (c-kit). An autocrine/paracrine mechanism has been proposed for the VEGF/KDR and SCF/ckit ligand receptor systems for human gastric cancers (Hassan et al., 1998; Takahashi et al., 1996; Yonemura et al., 1999). Mutation of the c-kit tyrosine kinase gene has been reported for gastrointestinal stromal tumors, and associated with poor clinical prognosis (Ernst et al., 1998; Sakurai et al., 1999). The trkA and hek5 receptor tyrosine kinases are expressed more frequently for welldierentiated gastric cancers than for the poorly dierentiated variants (Kiyokawa et al., 1994; Koizumi et al., 1998). While abl and src non-receptor tyrosine kinases are expressed in all gastric cell lines and cancer tissues examined, the src protein levels do not re¯ect activation status (kinase activity) (O'Neill et al., 1997; Takaishi et al., 2000). It is worth noting that H. pylori, an infectious pathogen, can up-regulate tyrosine kinase signal transduction pathways including src, EGFR, erbB2, MAPK and MKK4, following infection (Elitsur et al., 1999; Haruma et al., 2000; Meyer-Ter-Vehn et al., 2000; Naumann et al., 1999; Romano et al., 1998). Recently, H. pylori infection has been linked to peptic ulcer disease and gastric adenocarcinoma formation (EUROGAST Study Group, 1993; Segal et al., 1997; Kuniyasu et al., 2000). Reduced EGF and EGFR levels for stomach tissues have been observed following the eradication of H. pylori (Coyle et al., 1999), however, H. pylori status does not correlate with c-met and erbB2 immunoreactivities in clinicopathological analysis (Wu et al., 1997). Tyrosine kinase pro®les for human gastric cancer As has been discussed, about 15 ± 20 tyrosine kinases have been previously investigated for human gastric Oncogene


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Oncogene

cancers, with only c-met, k-sam, EGFR and erbB2 studied in greater detail. The past eorts were primarily directed toward selective types of tyrosine kinases, based on investigators' insights, expertise and reagents available. Considering the fact that tyrosine kinases often cross-talk and the activation of one may aect the other, there is a need to `see' the entire collection of expressed tyrosine kinases, if one is to fully appreciate the detailed oncogenic pathway(s) or to have the best chance of identifying tyrosine kinasebased biomarkers. Several high-through-put approaches including cDNA microarray (DeRisi et al., 1996; Moch et al., 1999), Serial Analysis of Gene Expression (Velculescu et al., 1995) and kinetic PCR (Kang et al., 1997) have been developed to study global gene expression pattern; the one that will be highlighted here is RT ± PCR based tyrosine kinase pro®ling approach, using degenerate primers that permit the ampli®cation of all tyrosine kinases which have these highly conserved motif in the catalytic domain (Robinson et al., 1996). In a single reaction, most, if not all expressed tyrosine kinases can be identi®ed. In this section, the results of a comparison of two matched pairs of gastric cancer and normal specimen are reviewed. In a later section, the results of a comparative study of four gastric cancer cell lines will be summarized. The latter results were obtained using an improved RT ± PCR based approach, tyrosine kinase display, which utilizes restriction digestions in lieu of cloning and sequencing, providing a simpler and more quantitative way of identi®cation of tyrosine kinases. Both approaches together have identi®ed a total of 44 tyrosine kinases expressed in various gastric cancers. Some of them have been previously reported to be expressed in gastric cancers, while others are somewhat unexpected to be found in gastric tissues. At least two of them were shown to be of prognostic values. As these primers also identify dual and a subset of serine/threonine kinases involved in signaling and cell cycle, information concerning their expressions is also included in the data summary. It is estimated that there are 1000 ± 2000 dierent protein kinases encoded by the human genome and about 10% of them represent tyrosine kinases (Hunter, 1987). A close scrutiny of the human genome database reveals the existence of close to 95 tyrosine kinases (see Robinson et al., this issue). With this number, it is reasonable to assume that about 30 ± 50 tyrosine kinases are expressed in a given cell, a number large enough to give a `®ngerprint' of a cancer cell, but small enough to be identi®ed in a simple screen. RT ± PCR approaches using degenerate primers have been employed by several laboratories to identify new tyrosine kinases for various human cells (for examples, Schultz and Nigg, 1993; Takahashi et al., 1995; Wilks, 1989), including gastric cancer cells (Kim et al., 1995). Robinson et al. (1996) designed a set of primers based on motifs in subdomain VII and IX of the catalytic domain of tyrosine kinases, and optimized the conditions, such that the reaction is of high ®delity (more than 90% of the amplicons are kinases) and broad speci®city (more than 90% of tyrosine kinases can be primed). Wu et al. (2000a) utilized a slight modi®cation of this approach (Lin et al., 1998, 1999a to compare two pairs of gastric cancers. The data summarized in Figure 1a reveals the expression of 32

dierent tyrosine kinases and a few dual kinases (mkk3, mkk4 etc) in these tissues, including 13 nonreceptor tyrosine kinases, 19 receptor tyrosine kinases. These studies con®rmed the earlier reports that c-met, k-sam and erbB2 are dierentially expressed in gastric cancer cells, but also identi®ed a number of other dierentially expressed tyrosine kinases that were previously unrecognized. The latter include fyn, itk, tyk2, c-fms, c-kit, cak, tie-1, mkk4which are more highly expressed in cancer tissues. The presence of cfms, c-kit and tie-1 is likely due to contaminating hematopoietic and vascular cells in the tissue specimens. Further immunohistochemical study however showed that tie-1, a receptor tyrosine kinase thought to have a restricted expression in endothelial and hematopoietic cells (Hashiyama et al., 1996; Sato et al., 1993), is actually expressed in gastric adenocarcinoma cells as well (Figure 1b). The aberrantly expressed tie-1 protein is localized in the cytoplasmic region of the cancer cells which are located on the luminal side of the adenocarcinoma tissues (Lin et al., 1999; Wu et al., 2000a). Moreover, the aberrant a

b

Figure 1 (a) Tyrosine kinase expression pro®les for human gastric cancer tissues. Two dierent pairs of gastric cancer surgical specimens were used for RT ± PCR ampli®cation of tyrosine kinases. Individual clones were randomly selected and their insert sequences were determined. A total of 243 tyrosine kinase clones were analysed from gastric cancer tissues and 129 clones from their matching normal gastric mucosa. The height of the bar indicates the number of clones identi®ed and sequenced. A total of 32 dierent tyrosine were identi®ed. (b) Immunohistochemical staining of tie-1 tyrosine kinase in human gastric adenocarcinoma sections. Anti-tie-1 protein antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) was used for immunohistochemical staining of human gastric cancer tissues by ABC methods (Lin et al., 1999) (original magni®cation 6400)


expression of tie-1 in gastric cancer cells was shown to be associated with reduced survival of the patients and serve as an independent predictor for survival (Lin et al., 1999). Likewise, expression of the mkk4 protein (a stress-induced dual kinase that phsophorylates the MAPK family of kinases) was observed in 44.8% (43/96) of gastric adenocarcinoma patients. Based on multivariate analysis, mkk4 was also shown to be an independent prognostic factor (relative risk=2.1) for gastric cancer progression (Wu et al., 2000a). A signi®cant role may be played by mkk4 kinase during the later stages of gastric cancer development. The tyrosine kinase pro®ling approach described above is eective for the identi®cation of nearly all tyrosine kinases expressed for particular cells or tissues. Yet, the cloning and sequencing steps remain tedious and the introduction of these steps compromises the quantitative aspects of this approach. A variation of this procedure utilizing restriction a

digestion analysis of the amplicon instead of cloning and sequencing was recently developed. This method, referred to as tyrosine kinase display in this review, combines the special features of the RT ± PCR/ degenerate-primers approach described above and the dierential display approach which separate individual genes by sizing on a sequencing gel. Brie¯y, by radio-labeling the 5' end of forward primers, subjecting the resulting amplicon to restriction digestions, and resolving the 5' end fragments in a sequencing gel, individual tyrosine kinases can be identi®ed by the characteristic sizes of their 5' end fragments. Since each molecule is labeled only at the 5' end, the relative intensity of the displayed fragments re¯ects the approximate molar ratio of the tyrosine kinase transcript copies. This approach which does away with cloning and sequencing should greatly improve the quantitative aspects of the procedure.

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b

Figure 2 (a) Tyrosine kinase display for the human gastric cancer cell line, AGS. Human gastric-cancer cell AGS cDNA was ampli®ed with degenerated primers (33P-label at the 5' end of the sense primer). Equal amounts of PCR amplicons (100 000 c.p.m.) were further digested with speci®c restriction endonucleases as indicated. The digested tyrosine kinase amplicon fragments were separated on a 7% denaturing polyacrylamide gel. Distinct bands representing dierent kinases were identi®ed according to a preestablished restriction-fragment database. (b) Tyrosine kinase expression pro®les for the four dierent human gastric cancer cell lines. The cDNA samples from four human gastric cancer cell lines (AGS, AZ521, HR, and KATO III) were subject to tyrosine kinase display analysis. Distinct tyrosine kinase bands were assigned by the sizes of their speci®c restriction fragments, and the expression level (radiation dose unit PSL) was measured with a Fuji BAS phospho-imager. The height of each bar indicates the PSL or the intensity of each band as measured by the phospho-imager. The upper closely-spaced bands are the intact amplicons belonging to kinases, which do not have the particular restriction site present in the ampli®ed region Oncogene


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A representative tyrosine kinase display example is shown in Figure 2a, the amplicons from a human gastric cancer cell line (AGS) were digested with seven dierent restriction enzymes and separated on a denaturing sequencing gel. Dierent tyrosine kinases and dual kinases were identi®ed according to their speci®c restriction-fragment sizes, when compared to a pre-established database. For example, the amplicon of hek5 (eph-B2), but not of any other tyrosine kinase, after Aci I digestion is predicted to yield a 5' end fragment of 74 bp in size and is identi®ed as such. Similarly, expressions of erbB2 and c-met genes in AGS cells are readily observed by Bsp12886 and Alu I digestion (Figure 2a). In some cases, fragments of a similar size represent a number of dierent kinases (labeled as `many'). Therefore, alternative restriction enzymes are necessary to ensure identi®cation of unique gene-speci®c restriction fragments. The intensity of individual bands visible in the phosphoimagerprocessed ®les can be used to quantify the expression level of the original transcripts. Thus, using a single RT ± PCR reaction and using a single gel, a tyrosine kinase pro®le can be obtained. The results are summarized in Figure 2b, with the height of the bar depicting the intensity of the band, and hence the expression level. In this ®gure, four dierent human gastric cancer cell lines (two American gastric cancer cell lines, AGS (Barranco et al., 1983) and HR (Shimizu et al., 1991), and two Japanese gastric cancer cell lines, AZ521 (Taki et al., 1985) and KATO III (Sekiguchi et al., 1978)) are compared. Thirty tyrosine kinases were identi®ed from these cell lines including 10 non-receptor tyrosine kinases and 20 receptor tyrosine kinases. The AGS line expressed the highest number of tyrosine kinases (25 dierent tyrosine kinases). All of the cell lines studied express yes, c-met, EGFR, erbB2 and k-sam at varying degrees, consistent with earlier reports. AZ521 express all four members of the erbB family of receptors. KATOIII expresses three of them (i.e., EGFR, erbB2 and erbB3) while the other two cell lines only harbor EGFR and erbB2. Ron, a receptor related to c-met, is expressed in two of the cell lines (AGS and KATO III). It is interesting to note that both members of the abl family of kinases, abl and arg, are expressed in all four cell lines. Most of these tyrosine kinases are expressed at moderate level (200 ± 5000 radiation dose units) with the exception of k-sam/

FGFR2 in KATO III and c-yes in HR, which are expressed 10 ± 100-fold higher than other kinases (above 20 000 radiation dose units). This high level of expression usually results from gene ampli®cation. This is indeed the case for k-sam in KATO III (Hara et al., 1998). Whether yes is ampli®ed at the genomic level in HR cells remains to be determined. Both HR and KATO III cells were from the metastases of gastric cancers. It has been demonstrated that k-sam is an important indicator for poorly dierentiated gastric cancer (Hattori et al., 1990). Whether yes or its related kinases involved in the progression of gastric cancer merits further investigation. Ampli®cation of yes oncogene was reported in a primary gastric carcinoma (Seki et al., 1985). Many tyrosine kinases are ubiquitously expressed in the various gastric cancer cell lines. The greatest variation in terms of the expression patterns seems to be associated with the eph/hek family of tyrosine kinases. For instance, hek5 is found to be expressed in all four cell lines with varying degree, consistent with its role in tumorigenesis (Kiyokawa et al., 1994). Additional two eph receptor tyrosine kinases were noted in the pro®le, with hek8 expressed for AGS and HR cells, while AZ521 and HR cells expressed hek11. For KATO III cells, neither hek8 nor hek11 were detected. This variation could mean that eph/hek family of kinases serve redundant functions or they may endow the dierent cell lines with dierent migratory and metastatic properties, as these kinases generally function in cell ± cell adhesion and communication (see Dodelet and Pasquale, this issue). The development of these tyrosine kinase pro®les provides a good framework to study the functions of these kinases in gastric carcinogenesis as well as to identify potential predictive markers and intervention targets.

Acknowledgments This study was supported in part by grants from Academia Sinica Medical Biotechnology Research and from the National Science Council to W-c Lin (NSC 90-2314-B001-007-M58) and US. NIH and DOD grants to HJ Kung. We would like to thank Mr Jyh-Shi Lin for his excellent technical assistance.

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