Overexpression of cortactin is involved in motility and metastasis of hepatocellular carcinoma

Journal of Hepatology 41 (2004) 629–636 www.elsevier.com/locate/jhep

Overexpression of cortactin is involved in motility and metastasis of hepatocellular carcinoma Makoto Chuma1,3, Michiie Sakamoto1,4, Jun Yasuda1, Gen Fujii1, Kazuaki Nakanishi1, Akira Tsuchiya1, Tsutomu Ohta2, Masahiro Asaka3, Setsuo Hirohashi1,* 1

Pathology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Genomics Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan 3 Department of Gastroenterology and Hematology, Hokkaido University, Sapporo, Japan 4 Department of Pathology, Keio University, School of Medicine, Tokyo, Japan

2

Background/Aims: The molecular basis of the metastasis of hepatocellular carcinoma (HCC) is not fully understood. The aim of this study was to elucidate the crucial genes involved in metastasis of HCC. Methods: We compared expression profiles among highly metastatic HCC cell lines and non-metastatic HCC cell lines by using oligonucleotide array to identify genes associated with metastasis. We further investigated the effect of identified gene on cell motility and metastasis in vitro and in vivo. Finally, we examined immunohistochemistry in human tissue samples. Results: We identified 39 genes whose expression levels were significantly correlated with metastatic ability (P!0.05). Of these genes, we further investigated cortactin, because this cortical actin-associated protein is a substrate of Src, whose activation has been shown to be involved in HCC cell migration and metastasis. Overexpression of cortactin in a non-metastatic HCC cell line increased cell motility, and resulted in metastasis in an orthotopic model. Furthermore, immunohistochemical expression of cortactin revealed its significant overexpression in HCC with intrahepatic metastasis compared with HCC without intrahepatic metastasis (P!0.005). Conclusions: Overexpression of cortactin may play a role in the metastasis of HCC by influencing cell motility, and cortactin could be a sensitive marker for HCC with intrahepatic metastasis. q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: Hepatocellular carcinoma; Intrahepatic metastasis; Cell motility; Cortactin; Oligonucleotide array; Short interfering RNA molecule 1. Introduction Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and one of the leading causes of cancer death in Japan [1]. Despite remarkable advances in diagnostic and therapeutic techniques, the prognosis of HCC patients remains poor because Received 28 January 2004; received in revised form 14 June 2004; accepted 22 June 2004; available online 10 July 2004 * Corresponding author. Tel.: C81-3-3542-2511; fax: C81-3-32482463. E-mail address: [email protected] (S. Hirohashi). Abbreviations: HCC, hepatocellular carcinoma; GAPDH, glyceraldehyde3-phosphate dehydrogenase; RT-PCR, reverse transcription polymerase chain reaction; siRNA, short interfering RNA molecule.

of the high incidence of hematogenous intrahepatic metastasis after initial treatment [2]. Hematogenous intrahepatic metastasis of HCC is observed frequently in advanced cases and is thought to develop through tumor cell dispersal via the portal vein [3]. In fact, vascular invasion of HCC is one of the most useful predictors of poor prognosis because it is accompanied by a high risk of recurrence [4,5]. Genetic alterations appear to be responsible for the development in HCC [6], however, the molecular mechanisms of hematogenous intrahepatic metastasis are far from clear. Understanding of intrahepatic metastasis at the molecular level is an important step toward the identification of predictive markers and more therapeutic targets for HCC recurrence. To analyze the mechanisms of intrahepatic metastasis, we previously

0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.06.018

630

M. Chuma et al. / Journal of Hepatology 41 (2004) 629–636

constructed metastatic models using orthotopic implantation of HCC cell lines [7]. Five HCC cell lines implanted orthotopically into SCID mice were found to form liver tumors: two of these cell lines, Li7 and KYN-2, resulted in hematogenous intrahepatic metastasis, whereas the other three, PLC/PRF/5, HepG2, and KIM-1, did not [7]. In this study, we first compared expression profiles among the two highly metastatic HCC cell lines and the three nonmetastastic HCC cell lines by using oligonucleotide array to identify the genes generally involved in intrahepatic metastasis. We showed that the molecular signatures were clearly different between highly metastatic HCC cell lines and non-metastastic HCC cell lines. Of these genes, we further investigated cortactin. The cortactin gene encodes a cytoplasmic actin-binding protein and facilitates the actin nucleation process, which is a critical step for actin polymerization [8,9] and consequential cell motility [10,11]. Cortactin is also a substrate of Src tyrosine kinase [12,13]. Because we previously showed involvement of c-Src in hepatocellular carcinoma cell migration [14] and a close correlation between cell motility and metastasis [7], we hypothesized that cortactin could play an important role in the intrahepatic metastasis of HCC. In this study, we overexpressed cortactin in a non-metastatic cell line, KIM1, or silenced cortactin by RNA interference in a highly metastatic cell line, KYN2, and examined the cell motility of each line. In addition, we investigated whether overexpression of cortactin could induce intrahepatic metastasis of KIM1 cells in the orthotopic model. Finally, to confirm the clinical significance of overexpression of cortactin, we examined the expression of cortactin by immunohistochemistry.

2. Materials and methods

of GAPDH in each sample was quantified by using the primer set 5 0 -GAAGGTGAAGGTCGGAGTC-3 0 (forward) and 5 0 -CCCGAATCACATTCTCCAAGAA-3 0 (reverse) and the amount of expression of cortactin was divided by that of the GAPDH in each sample. All PCR reactions were performed under the following conditions: 1 cycle at 50 8C for 2 min; 1 cycle at 95 8C for 10 min; then 40 cycles at 95 8C for 15 s, 55 8C for 30 s, and 72 8C for 1 min. Quantitative RT-PCR was performed at least three times.

2.4. Immunoblotting Cells were lysed and 5 mg total lysate was immunoblotted as described previously [17]. Primary antibody for cortactin (SC-11408; Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:2000 and antibody for alphatubulin at a dilution of 1:5000 (N356; Amersham Pharmacia Biotech, Buckinghamshire, England) were used to probe the blots.

2.5. Plasmids and transfection Human cortactin cDNA fragments containing whole open reading frames (1.7 kb) were amplified by PCR. The PCR products were cloned into the EcoR1-BamH1 site of the pIRESpuro vector (BD Biosciences Clontech, Palo Alto, CA). The cortactin expression vector and pIRESpuro without insert were independently transfected into the KIM1 cell using FuGENE 6 reagent (Roche, Indianapolis, IN), by following the manufacturer’s instructions. Stable transfectants were then selected by incubation at 37 8C with puromycin (Sigma-Aldrich, St. Louis, MO) for 2 weeks.

2.6. siRNA preparation and transfection The basic strategy for design of short interfering RNA molecule (siRNA) specific for cortactin was based on previous papers [18,19]. The siRNA sequences targeting cortactin were from position 5 0 -AATGATGTGAGTGA GAAGGAG-3 0 , as in the nucleotide sequence of cortactin. As a negative control, a siRNA was designed with the sequence 5 0 -GUUUCGAGGACUACUACAUUU-3 0 for sense and 5 0 -AUGUAGUAGUCCUCGAAACUU-3 0 for anti-sense. All siRNAs were purchased from Dharmacon Research (Lafayette, CO). For transient transfection of siRNAs, cells were plated at 1!105 cells per well in a six-well plate. Twenty-four hours after plating, the cells were transfected with a final concentration of 300 nmol/l of siRNA using Oligofectaminee reagent (Invitrogen) according to the manufacturers’ instructions. This procedure was carried out twice at an interval of 48 h, and after the second transfection the KYN2 cells were assayed.

2.1. Cell cultures and regents The human HCC cell lines PLC/PRF/5 and HepG2 were obtained from the American Type Culture Collection. KIM-1 and KYN-2 were kindly provided by Dr Masamichi Kojiro (Department of Pathology, Kurume University, Kurume, Japan), and Li7 was established in our laboratory [7].

2.2. RNA preparation and oligonucleotide array Total RNA was extracted from cell lines with Trizol reagent (Invitrogen Corp., Carlsbad, CA). Biotin-labeled cRNA was synthesized from 10 mg of total RNA by using a Super Script Choice System (Invitrogen) and hybridization of each cRNA to the probe array, HG-U95Av2 (Affymetrix, Santa Clara, CA) were performed as instructed by the manufacturer. Data analysis was done as described previously [15].

2.3. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis Real-time quantitative RT-PCR analysis was performed as reported previously [16]. The primer set 5 0 -TGA GTGTGTGTTCTTCCCCAAG-3 0 (forward) and 5 0 -CAC GTGACCTTCTGGAAAGACA-3 0 (reverse) was used for cortactin. For standardization of the amount of RNA, expression

2.7. Cell motility A cell motility HitKit (Cellomics, Pittsburgh, PA) with an Cellomics ArrayScan HCS system was used to analyze cell motility according to the manufacturers’ instructions. The assay is based on the visualization of trails of live cells plated on a lawn of microscopic fluorescent beads. Trypsinized cells in serum-free medium were plated on a 96-well microplate. After being cultured for 16 h, the cells were fixed with 4% paraformaldehyde and subjected to further analysis. All experiments were done in triplicate independently.

2.8. Orthotopic implantation in mice Orthotopic implantation of KIM1 cells and KIM1 transfectants was performed as described previously [7]. Mice were sacrificed 6 weeks after inoculation and autopsies were performed immediately. After macroscopic examination, the liver was removed and the number of tumors in each liver was counted and measured. Each liver sample was fixed in 10% formalin, embedded in paraffin and processed for histological examination. Intrahepatic metastatic lesions were defined as either: (1) lesions in lobes other than the lobe that was injected; or (2) lesions that were clearly separate from the primary tumor.

M. Chuma et al. / Journal of Hepatology 41 (2004) 629–636

2.9. Patients and tissue samples

3. Results

For immunohistochemical analysis, 152 progressed HCCs were analyzed. Sections were prepared from formalin-fixed, paraffin-embedded tissues of samples resected surgically between 1990 and 2002. Histological diagnosis was made according to WHO criteria [20,21]. The main clinicopathological features are presented in Table 1. HCCs with intrahepatic metastasis included tumor thrombus in the portal vein, because intrahepatic metastasis is thought to be generated via the portal venous system [22].

3.1. Two-way hierarchical clustering algorithm

2.10. Immunohistochemistry Immunohistochemical staining was done on formalin-fixed, paraffinembedded tissue sections by an immunoperoxidase method, as described previously [23]. Primary antibody for cortactin (SC-11408; Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500 was used for immunohistochemistry. Staining was evaluated by two independent observers. Cytoplasmic staining equal to or more intense than that of the endothelium and bile duct was considered positive.

2.11. Statistical analysis

631

To identify genes generally involved in intrahepatic metastasis, we compared the expression profiles of two highly metastatic HCC cell lines and three non-metastatic HCC cell lines. We filtered all genes, with the following limits: (1) presence (i.e. exactly expressed in the sample); (2) more than 2-fold increase or decrease of average difference between metastatic HCC cell lines and non-metastatic HCC cell lines; (3) Mann–Whitney U-test with significance set at P!0.05 to identify genes expressed differently between the two groups. In the 39 genes selected under the above criteria, a two-way hierarchical clustering algorithm successfully distinguished between 2 highly metastatic HCC cell lines and 3 nonmetastatic HCC cell lines (Fig. 1). 3.2. Cortactin expression in hepatocellular carcinoma cell line

Data are expressed as meansGSD. The level of cortactin mRNA in highly metastatic and non-metastatic lines was compared using unpaired t-test, and the area of cell motility was compared between the groups using paired t-test. The correlations between overexpression of cortactin protein and clinicopathological features were analyzed by the unpaired t-test and Fisher’s exact test. The ratio of metastasis in vivo was assessed using Fisher’s exact test. All statistical analyses were done using Stat View (Version 5.0) software (Abacus Concepts, Berkeley, CA). The results were judged significant at P!0.005.

From among the genes listed in Fig. 1, we next investigated cortactin, whose expression level was higher

Table 1 Immunohistochemical examination of cortactin in HCC Cortactin expression

Number of cases Mean ages (years)a Sex Male Female Virus maker HBs-Ag(C) HCV-Ab(C) NBNCb Maximal size (mm)a Differentiation Well Moderate Poor Intrahepatic metastasisc Present Absent a b c

(K)

(C)

88

64

63.0G10.1

61.9G9.8

59 29

42 22

27 49 12 41.6G19.3

17 38 9 47.8G23.0

19 46 23

9 27 28

26 62

40 24

P value

0.51 0.92

0.86

0.070 0.068

!0.005

MeanGSE. NBNC; patients without HBs-Ag(C) nor HCV-Ab(C). Included tumor thrombus in the portal vein.

Fig. 1. Two-way hierarchical clustering algorithm. Cluster map and phylogenetic tree resulted from a two-way, pairwise, average-linkage cluster analysis. Each color patch in the resulting visual map represents the expression level of the associated gene in that tissue sample, with a continuum of expression levels from blue (lowest) to bright red (highest). The two-way hierarchical clustering clustering algorithm successfully distinguished between two highly metastatic HCC cell lines and three non-metastatic lines. The scale bar reflects the fold increase (red) or decrease (blue) for any given gene relative to the median level of expression across all samples.

632

M. Chuma et al. / Journal of Hepatology 41 (2004) 629–636

Fig. 3. Western blot analysis was performed using human-specific cortactin antibody (each upper lanes) and the same amounts of proteins in a Western blot analysis using alpha-tubulin antibody (each lower lanes). (A) Expression of cortactin in 5 HCC cell lines. (B) KIM1 cells overexpressing wild-type cortactin (right), and mock transfectants (left). (C) KYN2 cells in which cortactin was silenced (right) and control cells (left).

these cell lines also showed similar expression pattern and confirmed the results by immunoblotting. 3.3. Expression of cortactin enhances cell motility

Fig. 2. Relationship between cortactin mRNA expression and cell motility in 5 HCC cell lines. (A) Real-time quantitative RT-PCR analysis. Cortactin mRNA expression levels in 5 HCC cell lines. Expression levels are normalized with GAPDH mRNA in each cell line. In the two highly metastatic liver cancer cell lines the average relative expression level was high (0.79G0.46) compared with that of the three non-metastatic (0.18G0.03), P!0.005. Bars, SE. (B) Comparison of motilities of 5 HCC cell lines in vitro. The proportion of motile cells in cultures of metastatic liver cancer cell lines was 6–10 times higher than in the three non-metastatic lines. Track area/cell area indicates cell motility.

in metastatic cell lines than in non-metastatic cell lines. To confirm this finding, we analyzed the level of cortactin mRNA by real-time quantitative RT-PCR. The average relative expression level of cortactin (cortactin/GAPDH) was significantly higher in metastatic lines than in nonmetastatic lines (0.79G0.46 vs. 0.18G0.03; P!0.005), although Li7 expressed less cortactin than the KYN2 line (Fig. 2A). Next, we used an antibody against cortactin to investigate protein expression of cortactin in 5 HCC cell lines. As in the RT-PCR analysis, the KYN2 line showed significantly higher expression of cortactin than the Li7 line and the three non-metastatic cell lines (Fig. 3A). Immunohistochemical expression of cortactin in orthotopic tumor of

Although there was a slight discrepancy between the level of cortactin mRNA and protein in Li7, the close correlation between the cortactin mRNA level and cell motility (Fig. 2B) encouraged us to investigate whether overexpression or silencing of cortactin was involved in the motility of human HCC cell lines. Human wild-type (wt) cortactin was overexpressed in one non-metastatic cell line, KIM1, in which the expression of cortactin mRNA was lowest among the 5 HCC cell lines investigated. Following puromycin selection, one bulk culture was prepared and overexpression of cortactin was confirmed by immunoblotting (Fig. 3B). Further, we silenced cortactin in KYN2 cells by RNA interference. Cortactin was efficiently and specifically silenced 48 h post-transfection of siRNA, as shown by immunoblotting (Fig. 3C). As reported previously [6], the proportion of motile cells in cultures of KYN2 cells was normally highest, whereas that in KIM1 cells was normally lowest (Fig. 2B). KIM1 cells overexpressing wt-cortactin were nearly three times as motile as mock transfectants (Fig. 4A and C; P!0.005). On the other hand, KYN2 cells in which cortactin had been silenced were less than half as motile as control cells (Fig. 4B and D; P!0.005). 3.4. Overexpression of cortactin potentiates intrahepatic metastasis in hepatocellular carcinoma cell lines in vivo First, we examined the cell growth rates of KIM1 wt-cortactin transfectants and mock transfectants in vitro, and there was no significant difference between them

M. Chuma et al. / Journal of Hepatology 41 (2004) 629–636

633

Fig. 5. Analysis of the effect of cortactin on the growth of KIM1 cells. (A) KIM1 cells wt-cortactin transfectants versus mock transfectants were plated on a 12-well plate at a density of 0.4!105 cells/well in RPMI supplemented with 10% FBS. On days 1–6, cells were harvested and counted with a hemocytometer. Data are the mean of 4 independent experiments. (B) Effect of cortactin on tumorigenecity in the orthotopic implantation model. Tumorigenecity was evaluated from the maximum tumor diameter. Bars, SD.

3.5. Protein expression of cortactin in hepatocellular carcinoma tissues

Fig. 4. Expression of cortactin enhances cell motility. HCC cell lines were plated on fibronectin-coated dishes, allowed to adhere and spread for 16 h, and the results recorded by Array Scan. Track area/cell area indicates cell motility. (A, C) Mean motility of cortactin-overexpressing KIM1 cells (3.66G0.40) was nearly three times greater than that of mock transfectant (1.31G0.08). (B, D) Mean motility of cortactinsilenced KYN2 cells (4.76G0.57) was less than half as great as that of control cells (9.35G0.98). Blue lines indicate track area and red lines indicate cell area. Bars, SD. The track area is proportional to the magnitude of cell movement.

(Fig. 5A). Then we investigated the effect of cortactin on hematogenous intrahepatic metastasis of KIM1 cells in vivo, as reported previously [7]. The maximum primary tumor diameter was not significantly different between wt-cortactin transfectants and mock transfectants (Fig. 5B), suggesting that overexpression of cortactin did not affect primary tumor growth in vivo, or in vitro. However, the incidence of hematogenous intrahepatic metastasis was promoted in wt-cortactin transfectants compared to mock transfectants (Fig. 6; Table 2). Though the difference was not statistically significant (P!0.27), it was suggested that overexpression of cortactin promoted metastasis to some extent by increasing cell motility without any change in growth.

To determine whether cortactin is also overexpressed at the protein level in human HCC tissues and involved in intrahepatic metastasis, we employed an antibody against cortactin in an immunohistochemical study (Fig. 7). Hepatocytes in non-cancerous liver tissues with chronic hepatitis or cirrhosis showed no immunostaining or only focal and faint staining in the cytoplasm. However, the endothelium and bile duct always stained strongly and thus served as an internal control of positive staining (Fig. 7B and D). Some sinusoidal cells also showed moderate staining. Some HCC specimens without intrahepatic metastasis showed little cortactin immunoreactivity (Fig. 7B), whereas HCC specimens with intrahepatic metastasis showed strong cortactin immunoreactivity (Fig. 7D and F). Strong immunoreactivity was observed in the cell membrane and cytoplasm (Fig. 7), and in most cases the intensity of staining of the cytoplasm corresponded to that of the cell membrane. To evaluate the relation between cortactin and clinicopathological features were judged positive if strong cortactin expression was observed in more than 20% of tumor cells and negative if cortactin expression was lower than 20%. Table 1 shows that intrahepatic metastasis were significantly associated with cortactin expression (P!0.005).

4. Discussion The recent development of cDNA microarray or oligonucleotide array technology, a high-throughput method of monitoring gene expression, has made it possible

634

M. Chuma et al. / Journal of Hepatology 41 (2004) 629–636

Fig. 6. Growth and hematogenous intrahepatic metastasis of overexpressed cortactin in KIM1 cells in SCID mice after orthotopic implantation. (A) Macroscopic view of KIM1 tumor. Arrowhead marks the primary tumor developed at the injected lobe, and arrows mark intrahepatic metastases in the non-injected lobe. (B) Histological findings of KIM1 tumor thrombi in the portal vein (hematoxylin-eosin stain, original magnification !40).

to analyze the expression of thousands of genes at once [24,25]. Consequently, new classifications of cancers can now be proposed on the basis of the altered expression of multiple genes in tumor tissues [24–28]. To elucidate the characteristic changes associated with intrahepatic metastasis, we globally analyzed the gene expression of two highly metastatic HCC cell lines and three non-metastatic HCC cell lines by using oligonucleotide array. We showed that a two-way hierarchical clustering algorithm successfully distinguished between two highly metastatic HCC cell lines and three non-metastatic HCC cell lines. These results

Fig. 7. Immunohistochemical localization of cortactin in HCC. Representative histology of HCC (A, C, E) (hematoxylin-eosin stain, original magnification, !20), and cortactin immunostaining of each serial section (B, D, F). Portal vein epithelium (arrow) and bile duct (arrowhead) served as the positive control. Non-cancerous liver (N) in B, D, F and HCC (T) without intrahepatic metastasis (B) showed no cortactin immunoreactivity. HCC (T) with intrahepatic metastasis in D and F showed strong cortactin immunoreactivity (D, 50%; F, 80%).

indicate that there is a clear difference in molecular signatures between highly metastatic HCC cell lines and non-metastatic HCC cell lines, and identified 39 genes whose expression levels were significantly correlated with metastatic ability. Cancer metastasis is a multi-step process that involves cell detachment from the primary tumor; entry into the vascular or lymphatic system; dispersal through the circulation; and proliferation following extravasation in the target organs [29]. Many studies have shown the importance and mechanisms of invasion, one of the first steps in metastasis. Loss of cell–cell contact caused by downregulation of Ecadherin and increased cell motility have been reported to be critical steps in this process [7,30,31]. Our previous report [14] and others [32] determined that c-Src activation is critically involved in carcinoma cell migration

Table 2 Hematogenous intrahepatic metastasis of wt-cortactin transfectants of KIM1 cells No. of mice with intrahepatic metastasis Macroscopic

Mock Bulk

Microscopic

Injected lobe

Non-injected lobe

Total

Injected lobe

Non-injected lobe

Total

0/10 2/15

0/10 1/15

0/10 3/15

0/10 3/15

0/10 1/15

0/10 4/15

The data represent the number of mice with hematogenous intrahepatic metastasis over the total number of mice evaluated.

M. Chuma et al. / Journal of Hepatology 41 (2004) 629–636

and metastasis. Therefore, we selected cortactin, a substrate of Src, from 39 genes and examined whether overexpression of cortactin was involved in cell motility and intrahepatic metastasis of human HCC cell lines. We demonstrated that overexpression of cortactin in a non-metastatic HCC cell line increased cell motility and silencing of cortactin in a metastatic HCC cell line reduced cell motility without growth change, and transfection of non-metastatic HCC cells with wt-cortactin resulted in metastasis in vivo to some extent. Furthermore, in human tissue samples, immunohistochemical examination of cortactin revealed its significant overexpression in HCC with intrahepatic metastasis compared with HCC without intrahepatic metastasis. The relatively mild effect of cortactin overexpression on cancer metastasis in vivo could be explained by the fact that although cortactin is necessary, it is insufficient alone for completion of the multi-step metastasis process. Cortactin, a p80/85 protein first identified as a Src kinase substrate [12,13] is thought to be involved in cytoskeletal reorganization [8,9] and cell adhesion [12,33]. The cortactin gene, EMS1, maps to chromosome 11q13, a region amplified in several cancers [34–37]. The amplification or overexpression of cortactin in head, neck, and breast carcinomas is closely associated with poor prognosis [34,35] Although Yuan [37] recently described cortactin amplification in human HCC tissues and overexpression in human HCC cell lines, to our knowledge no reports have described cortactin overexpression in human HCC tissues. Overexpression of cortactin in human HCC tissues is novel, and cortactin could be a new predictive marker for HCC with intrahepatic metastasis. The mechanism by which overexpression of cortactin facilitates the interaction of tumor cells is not clear. Recently, it has become clear that cortactin is involved in the regulation of cell motility and invasion [10,11,38,39]. Some reports have demonstrated that overexpression of cortactin in NIH3T3 cells results in increased cell motility and invasiveness without changing growth rate or morphology [10], and another report demonstrated that cortactin promotes bone metastasis of breast cancer cells [38]. Therefore, it is possible that cortactin acts as a signaling molecule in the regulation of the dynamics of the actin cytoskeleton, and that cortactin overexpression is closely associated with cell motility and may play a role in intrahepatic metastasis. The role of cortactin in cytoskeletal reorganization is further highlighted by the recent finding that cortactin binds to the Arp2/ 3 complex and activates Arp2/3-complex-mediated actin polymerization [40,41]. Interestingly, Arp2/3 was also overexpressed in metastatic cell lines compared with nonmetastatic cell lines in our present study, suggesting that co-overexpression and interaction might be important in motility and metastasis. In addition to the gene for cortactin, we were able to identify several candidate genes involved in intrahepatic metastasis. Some of the up-regulated genes have previously been correlated with tumor malignancy and metastasis in

635

several types of cancers. For example, Pim-1 oncoprotein is a serine/threonine kinase that can closely cooperate with c-Myc in lymphomagenesis [42] and is significantly correlated with clinical outcome in prostate cancer [43]. Nup88, an 88-kd nucleoporin found to be associated in a dynamic subcomplex with the oncogenic nucleoporin CAN/Nup 214 [44] is involved in progression from primary to metastatic melanomas [45] and its expression is positively related to distant metastasis of colorectal cancer [46]. Overexpression of hepatocyte nuclear factor 3 has been described in HCC [47]. In conclusion, although the precise mechanism remains to be elucidated, overexpression of cortactin was closely associated with intrahepatic metastasis in human HCC and was a sensitive marker for HCC with intrahepatic metastasis. It could be a new predictive marker and a new therapeutic target of HCC metastasis.

Acknowledgements This study was supported by a Granting-Aid for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan, and for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology, and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research. M.C. is a recipient of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research.

References [1] Okuda K. Hepatocellular carcinoma: recent progress. Hepatology 1992;15:948–963. [2] Nagao T, Inoue S, Yoshimi F, Sodeyama M, Omori Y, Mizuta T, et al. Postoperative recurrence of hepatocellular carcinoma. Ann Surg 1990;211:28–33. [3] Yamanaka N, Okamoto E, Fujihara S, Kato T, Fujimoto J, Oriyama T, et al. Do the tumor cells of hepatocellular carcinomas dislodge into the portal venous stream during hepatic resection?. Cancer 1992;70: 2263–2267. [4] Okada S, Shimada K, Yamamoto J, Takayama T, Kosuge T, Yamasaki S, et al. Predictive factors for postoperative recurrence of hepatocellular carcinoma. Gastroenterology 1994;106:1618–1624. [5] Shirabe K, Kanematsu T, Matsumata T, Adachi E, Akazawa K, Sugimachi K. Factors linked to early recurrence of small hepatocellular carcinoma after hepatectomy: univariate and multivariate analyses. Hepatology 1991;14:802–805. [6] Laurent PP, Legoix P, Bluteau O, Belghiti J, Franco D, Binot F, et al. Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis. Gastroenterology 2001; 120:1763–1773. [7] Genda T, Sakamoto M, Ichida T, Asakura H, Kojiro M, Narumiya S, et al. Cell motility mediated by rho and Rho-associated protein kinase plays a critical role in intrahepatic metastasis of human hepatocellular carcinoma. Hepatology 1999;30:1027–1036.

636

M. Chuma et al. / Journal of Hepatology 41 (2004) 629–636

[8] Weed SA, Parsons JT. Cortactin: coupling membrane dynamics to cortical actin assembly. Oncogene 2001;20:6418–6434. [9] Wu H, Parsons JT. Cortactin, an 80/85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex. J Cell Biol 1993;120:1417–1426. [10] Patel AS, Schechter GL, Wasilenko WJ, Somers KD. Overexpression of EMS1/cortactin in NIH3T3 broblasts causes increased cell motility and invasion in vitro. Oncogene 1998;16:3227–3232. [11] Huang C, Liu J, Haudenschild CC, Zhan X. The role of tyrosine phosphorylation of cortactin in the locomotion of endothelial cells. J Biol Chem 1998;273:25770–25776. [12] Schuuring E, Verhoeven E, Litvinov S, Michalides RJ. The product of the EMS1 gene, amplified and overexpressed in human carcinomas, is homologous to a v-src substrate and is located in cell-substratum contact sites. Mol Cell Biol 1993;5:2891–2898. [13] Huang C, Ni Y, Wang T, Gao Y, Haudenschild CC, Zhan X. Downregulation of the filamentous actin cross-linking activity of cortactin by Src-mediated tyrosine phosphorylation. J Biol Chem 1997;272: 13911–13915. [14] Sakamoto M, Takamura M, Ino Y, Miura A, Genda T, Hirohashi S. Involvement of c-Src in carcinoma cell motility and metastasis. Jpn J Cancer Res 2001;92:941–946. [15] Chuma M, Sakamoto M, Yamazaki K, Ohta T, Ohki M, Asaka M, et al. Expression profiling in multistage hepatocarcinogenesis: identification of HSP70 as a molecular marker of early hepatocellular carcinoma. Hepatology 2003;37:198–207. [16] Kanai Y, Ushijima S, Nakanishi Y, Hirohashi S. Reduced mRNA expression of the DNA demethylase, MBD2, in human colorectal and stomach cancers. Biochem Biophys Res Commun 1999;264:962–966. [17] Nakanishi K, Sakamoto M, Yasuda J, Takamura M, Fujita N, Tsuruo T, et al. Critical involvement of the phosphatidylinositol 3-kinase/Akt pathway in anchorage-independent growth and hematogeneous intrahepatic metastasis of liver cancer. Cancer Res 2002;62: 2971–2975. [18] Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494–498. [19] Harborth J, Elbashir SM, Bechert K, Tuschl T, Weber K. Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Sci 2001;114:4557–4565. [20] Hirohashi S, Ishak KG, Kojiro M, Wanless IR, Theise ND, Tsukuma H, et al. Hepatocellular carcinoma. In: Hamilton SR, Aaltonen LA, editors. Pathology and genetics of tumors of the digestive system. Lyon: IARC Press; 2000, p. 159–172. [21] Ishak KG, Anthony PP, Sobin LH. Histological typing of tumors of the liver. New York: World Health Organization; 1994. [22] Osada T, Sakamoto M, Ino Y, Iwamatsu A, Matsuno Y, Muto T, et al. E-cadherin is involved in the intrahepatic metastasis of hepatocellular carcinoma. Hepatology 1996;24:1460–1467. [23] Hsu SM, Raine L, Fanger H. Use of avidin–biotin–peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577–580. [24] Schena M, Shalon D, Davis RW, Brown PO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995;270:467–470. [25] Lockhart DJ, Dong H, Byrne MC, Follettie MT, Gallo MV, Chee MS, et al. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat Biotechnol 1996;14:1675–1680. [26] Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000;403:503–511. [27] Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov JP, et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 1999; 286:531–537.

[28] Bittner M, Meltzer P, Chen Y, Jiang Y, Seftor E, Hendrix M, et al. Molecular classification of cutaneous malignant melanoma by gene expression profiling. Nature 2000;406:536–540. [29] Poste G, Fidler IJ. The pathogenesis of cancer metastasis. Nature 1980;283:139–146. [30] Hirohashi S. Inactivation of the E-cadherin-mediated cell adhesion system in human cancers. Am J Pathol 1998;153:333–339. [31] Takamura M, Sakamoto M, Genda T, Ichida T, Asakura H, Hirohashi S. Inhibition of intrahepatic metastasis of human hepatocellular carcinoma by Rho-associated protein kinase inhibitor Y-27632. Hepatology 2001;33:577–581. [32] Windham TC, Parikh NU, Siwak DR, Summy JM, McConkey DJ, Kraker AJ, et al. Src activation regulates anoikis in human colon tumor cell lines. Oncogene 2002;21:7797–7807. [33] Vuori K, Ruoslahti E. Tyrosine phosphorylation of p130Cas and cortactin accompanies integrin-mediated cell adhesion to extracellular matrix. J Biol Chem 1995;270:22259–22262. [34] Rodrigo JP, Garcia LA, Ramos S, Lazo PS, Suarez C. EMS1 gene amplification correlates with poor prognosis in squamous cell carcinomas of the head and neck. Clin Cancer Res 2000;8: 3177–3182. [35] Hui R, Ball JR, Macmillan RD, Kenny FS, Prall OWJ, Campbell DH, et al. EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival. Oncogene 1998;17:1053–1059. [36] Bringuier PP, Tamimi Y, Schuuring E, Schalken J. Expression of cyclin D1 and EMS1 in bladder tumors; relationship with chromosome 11q13 amplification. Oncogene 1996;12:1747–1753. [37] Yuan BZ, Zhou X, Zimonjic DB, Durkin ME, Popescu NC. Amplification and overexpression of the EMS 1 oncogene, a possible prognostic marker, in human hepatocellular carcinoma. J Mol Diagn 2003;1:48–53. [38] Li Y, Tondravi M, Liu J, Smith E, Haudenschild CC, Kaczmarek M, et al. Cortactin potentiates bone metastasis of breast cancer cells. Cancer Res 2001;61:6906–6911. [39] Bowden ET, Barth M, Thomas D, Glazer RI, Mueller SC. An invasion-related complex of cortactin, paxillin and PKCmu associates with invadopodia at sites of extracellular matrix degradation. Oncogene 1999;18:4440–4449. [40] Uruno T, Liu J, Zhang P, Fan Y, Egile C, Li R, et al. Activation of Arp2/3 complex-mediated actin polymerization by cortactin. Nat Cell Biol 2001;3:259–266. [41] Weed SA, Karginov AV, Schafer DA, Weaver AM, Kinley AW, Cooper JA, et al. Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex. J Cell Biol 2000;151:29–40. [42] Mochizuki T, Kitanaka C, Noguchi K, Sugiyama A, Kagaya S, Chi S, et al. Pim-1 kinase stimulates c-Myc-mediated death signaling upstream of caspase-3 (CPP32)-like protease activation. Oncogene 1997;12:1471–1480. [43] Dhanasekaran SM, Barrette TR, Ghosh D, Shah R, Varambally S, Kurachi K, et al. Delineation of prognostic biomarkers in prostate cancer. Nature 2001;412:822–826. [44] Martinez N, Alonso A, Moragues MD, Ponton J, Schneider J. The nuclear pore complex protein Nup88 is overexpressed in tumor cells. Cancer Res 1999;59:5408–5411. [45] Zhang H, Schneider J, Rosdahl I. Expression of p16, p27, p53, p73 and Nup88 proteins in matched primary and metastatic melanoma cells. Int J Oncol 2002;1:43–48. [46] Emterling A, Skoglund J, Arbman G, Schneider J, Evertsson S, Carstensen J, et al. Clinicopathological significance of Nup88 expression in patients with colorectal cancer. Oncology 2003;64: 361–369. [47] Xu L, Hui L, Wang S, Gong J, Jin Y, Wang Y, et al. Expression profiling suggested a regulatory role of liver-enriched transcription factors in human hepatocellular carcinoma. Cancer Res 2001;61: 3176–3181.