Oncogene (2003) 22, 4656–4663 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Downregulation of stomach cancer-associated protein tyrosine phosphatase-1 (SAP-1) in advanced human hepatocellular carcinoma Hidenobu Nagano1,2, Tetsuya Noguchi*,1, Kenjiro Inagaki1, Seitetsu Yoon1, Takashi Matozaki3, Hiroshi Itoh2, Masato Kasuga1 and Yoshitake Hayashi2 1Division of Diabetes, Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan; 2Division of Surgical Pathology, Department of Biomedical Informatics, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan; 3Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan SAP-1 (stomach cancer-associated protein tyrosine phos- phosphorylation of cellular targets of these enzymes phatase-1) is a transmembrane-type protein tyrosine (Cantley et al., 1991; Fantl et al., 1993). Protein tyrosine phosphatase that has been implicated as a negative phosphatases (PTPs) likely also affect carcinogenesis, regulator of integrin-mediated signaling. The potential given that they counteract the action of protein tyrosine role of this enzyme in hepatocarcinogenesis has now been kinases (Tonks, 1993; Walton and Dixon, 1993; Hunter, investigated by examining its expression in 32 surgically 1995). Indeed, certain PTPs have been associated with excised human hepatocellular carcinoma (HCC) speci- the development of some types of cancer. For example, mens. Both immunohistochemical and immunoblot ana- the expression of HPTPb is decreased in many lung lyses revealed that normal liver tissue, as well as tissue cancers (De Vries et al., 1991), and the gene encoding affected by chronic hepatitis or cirrhosis, contained PTPg is frequently deleted in both renal and lung substantial amounts of SAP-1. The expression level of cancers (LaForgia et al., 1991; Tsukamoto et al., SAP-1 in 75% of well-differentiated HCCs was similar to 1992). or higher than that observed in the surrounding non- Human hepatocellular carcinoma (HCC) has been cancerous tissue. In contrast, the abundance of SAP-1 in thought to develop as a consequence of combinations of 85.7% of moderately differentiated HCCs and in all abnormalities in various genes such as those encoding poorly differentiated HCCs was greatly reduced com- Rb, p53, p16, and telomerase (Rogler and Chisari, 1992; pared with that in the adjacent tissue. Indeed, SAP-1 was Nishida et al., 1993; Tahara et al., 1995). As in other cell almost undetectable in 83.3% of poorly differentiated types, the tyrosine phosphorylation of cellular proteins HCCs. Furthermore, expression of recombinant SAP-1 in is indispensable for the growth and differentiation of two highly motile human HCC cell lines resulted in a hepatocytes (Palmer et al., 1999). In addition, the change in morphology and a marked reduction in both expression of several PTPs is upregulated during migratory activity and growth rate. In conclusion, these regeneration of the liver after partial hepatectomy in results indicate that SAP-1 expression is downregulated mice and rats (Higashitsuji et al., 1995; Kitamura et al., during the dedifferentiation of human HCC, and that this 1995; Saito et al., 1996). These observations thus downregulation may play a causal role in disease implicate tyrosine phosphorylation events and, hence, progression. PTPs in hepatocellular function under both physiologi- Oncogene (2003) 22, 4656–4663. doi:10.1038/sj.onc.1206588 cal and pathological conditions. Dysfunction of PTPs may thus contribute to the development of HCC, Keywords: tyrosine phosphatase; cell migration; de- although few studies to date have implicated specific differentiation; hepatoma PTPs in the pathogenesis of this disease (Ikuta et al., 1994; Kitamura et al., 1995). SAP-1 (stomach cancer-associated protein tyrosine phosphatase-1) was first identified as a transmembrane- Introduction type (receptor-like) PTP that is abundant in a subset of pancreatic and colorectal cancer cell lines (Matozaki Many genetic alterations in both oncogenes and tumor et al., 1994). This enzyme belongs to the class 2 suppressor genes contribute to multistep carcinogenesis subfamily of receptor-like PTPs and is structurally in humans (Sugimura, 1992). The fact that several similar to HPTPb, DEP-1, and DPTP10D (Krueger oncogene products, including Bcr-Abl and v-Src, et al., 1990; Tian et al., 1991; Ostman et al., 1994). We possess high protein tyrosine kinase activity suggests have recently shown that SAP-1 mediates the depho- that carcinogenesis is promoted by the aberrant sphorylation of several focal adhesion-associated sub- strates and, consequently, inhibits cell proliferation by *Correspondence: T Noguchi; E-mail: [email protected] suppressing growth factor-elicited mitogenic signaling as Received 5 August 2002; revised 17 March 2003; accepted 17 March 2003 well as by inducing apoptotic cell death (Noguchi et al., Tyrosine phosphatase SAP-1 in human hepatocellular carcinoma H Nagano et al 4657 2001; Takada et al., 2002). The enzymatic activity of Table 1 Clinical characteristics of the study subjects SAP-1 is also increased in cells that exhibit density- WD-HCC MD-HCC PD-HCC dependent growth arrest (Noguchi et al., 2001). Characteristic (n ¼ 12) (n ¼ 14) (n ¼ 6) Furthermore, the abundance of SAP-1 mRNA increases during differentiation of breast cancer cells in response Sex (male/female) 12/0 12/2 4/2 Mean (range) age (years) 58.8 (52-67) 56.3 (34-72) 65.0 (63-66) to anticancer drugs (Keane et al., 1996). These observa- HBsAg/HCV Ab/alcohol 2/10/0 6/8/0 0/5/1 tions suggest that SAP-1 might act as a tumor Serum AFP (ng/ml) 4-303 21-19,840 5117-313,930 suppressor by generating a signal that results in growth Histological background arrest and differentiation of cancer cells. However, the Chronic hepatitis (n)2 0 0 potential roles of this PTP in carcinogenesis have Liver cirrhosis (n)10146 remained unclear. The differentiation status of each tumor was classified into three SAP-1 is abundant in the liver (Matozaki et al., 1994) categories according to the criteria of Edmondson and Steiner: well- and might therefore contribute to the initiation or differentiated (WD) HCC, corresponding to grades I to II; moderately progression of HCC. To investigate this hypothesis, differentiated (MD) HCC, corresponding to grades II or III; and we have now examined the expression of SAP-1 in poorly differentiated (PD) HCC, corresponding to grades III or IV. Disease etiology was classified according to serum positivity for human HCCs at various stages of differentiation. hepatitis B virus surface antigen (HBsAg) or for antibodies to hepatitis Our results show that the abundance of SAP-1 is C virus (HCVAb), or to alcohol intake. AFP, a-fetoprotein inversely related to the aggressiveness of HCC. Further- more, we show that forced expression of SAP-1 in highly motile HCC cells resulted in a morphological change that was accompanied by a marked reduction in (Figure 2a). Consistent with the immunohistochemical cell migration. data, the amount of SAP-1 in WD-HCC tissue was greater than that in MD-HCC or PD-HCC (Figure 2b). In contrast, the expression of SAP-1 in the surrounding Results cirrhotic tissue of these different tumors appeared similar (Figure 2b). The relation between the level of SAP-1 expression Expression of SAP-1 in human HCCs and the differentiation status of HCC was then analysed HCC specimens from 32 individuals were examined for statistically. Among the three stages of HCC differentia- SAP-1 expression by immunohistochemical analysis tion, the abundance of SAP-1 decreased in the rank with two different antibodies specific for this protein. order WD-HCC4MD-HCC4PD-HCC (Po0.01 for The clinical characteristics of the patients are provided WD-HCC versus MD-HCC and for MD-HCC versus in Table 1, and representative immunohistochemical PD-HCC) (Figure 3). Furthermore, the amount of SAP- data are shown in Figure 1. In normal hepatocytes, 1 in either MD-HCC or PD-HCC tissue was signifi- SAP-1 was detected predominantly at the cell membrane cantly smaller than that in the respective noncancerous and to a lesser extent in the cytoplasm (Figure 1a, e). tissue, with this reduction being more pronounced for Immunoreactivity was not apparent in sinusoidal PD-HCC (Figure 3). The abundance of SAP-1 in endothelial cells, whereas epithelial cells of the bile duct adjacent noncancerous tissue did not differ significantly yielded a moderate signal (Figure 1a, e). The endothe- among individuals with WD-HCC, with MD-HCC, or lium of the portal tract vessels was weakly positive for with PD-HCC. Given that human HCC progresses SAP-1, but mesenchymal cells were negative (Figure 1a, through dedifferentiation of the tumor, these results e). Nine of 12 (75%) well-differentiated (WD) HCCs indicate that the level of SAP-1 expression is inversely expressed SAP-1 at a level similar to or higher than that related to tumor aggressiveness. apparent in the surrounding noncancerous tissue, although the remaining three WD-HCCs also expressed Effects of ectopic expression of SAP-1 on HCC cell a substantial amount of this protein (Figure 1b, f and morphology and migration Table 2). In 12 of 14 (85.7%) specimens of moderately differentiated (MD) HCC, the abundance of SAP-1 in A reduced abundance of SAP-1 might contribute to the the cancerous tissue was substantially lower than that in acquired characteristics of advanced HCC such as the the adjacent noncancerous tissue (Figure 1c, g and abilities to metastasize and to invade tissue. Alterna- Table 2). All six poorly differentiated (PD) HCCs tively, it might be a consequence of disease progression. contained much smaller amounts of SAP-1 than did To distinguish between these possibilities, we transfected the surrounding noncancerous tissue (Figure 1d, h HLF and HLE cells, both of which are highly motile and Table 2); indeed, SAP-1 was almost undetectable and were derived from human PD-HCC (Doi et al., in five of six (83.3%) PD-HCCs (Table 2).