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Human Cancer Biology |
Authors' Affiliations: 1 Genome Science Division and 2 Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, the University of Tokyo; 3 Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine, the University of Tokyo; 4 Perseus Proteomics, Inc., Tokyo, Japan; and 5 Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Shizuoka, Japan; 6 Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd., Kanagawa, Japan; and 7 Department of Pathology and 8 Department of Gastroenterology, Yokohama City University Graduate School of Medicine, Yokohama, Japan
Requests for reprints: Hiroyuki Aburatani, Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan. Phone: 81-3-5452-5352; Fax: 81-3-5452-5355; E-mail: haburata-tky{at}umin.ac.jp.
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Abstract |
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Experimental Design: Gene expression profiles of pooled total RNA from three tissues each of moderately differentiated and poorly differentiated hepatocellular carcinoma were compared with those of normal liver, noncancerous liver tissue in hepatocellular carcinoma patients, 30 normal tissue samples, and five fetal tissue samples. Target genes up-regulated specifically in hepatocellular carcinoma were validated by immunohistochemical analysis and complement-dependent cytotoxicity assay using monoclonal antibodies generated against target molecules.
Results: The human homologue of the Drosophila Roundabout gene, axon guidance receptor homologue 1, ROBO1/DUTT1, a member of the immunoglobulin superfamily, was highly expressed in hepatocellular carcinoma, whereas it showed only a limited distribution in normal tissues. On immunohistochemical analysis using a newly generated anti-ROBO1 monoclonal antibody, positive signals were observed in 83 of 98 cases of hepatocellular carcinoma (84.7%). The mAb B2318C induced complement-dependent cytotoxicity in ROBO1-expressing cell lines and in the liver cancer cell line PLC/PRF/5. Strikingly, the ectodomain of ROBO1 was detected not only in the culture medium of liver cancer cell lines (PLC/PRF/5, HepG2, etc.) but also in sera from hepatocellular carcinoma patients (6 of 11).
Conclusions: This is the first report that ROBO1 is overexpressed in hepatocellular carcinoma and shed into serum in humans. These observations suggest that ROBO1 is a potential new serologic marker for hepatocellular carcinoma and may represent a new therapeutic target.
Targeted therapies are dramatically changing the treatment modalities for breast, lung, and colon cancers. In breast cancer therapy, trastuzumab (Herceptin), a humanized recombinant monoclonal antibody (mAb) that recognizes the extracellular domain of HER2 transmembrane protein, is among the first immunologic target-specific drugs that have been licensed for clinical use, and its development represents a model for the integration of new agents with classic treatment strategies (2, 3). Therefore, the identification of tumor-associated cell surface antigens is critical in the development of tumor-targeted antibody therapy (3).
The ideal expression pattern of a cancer-specific target antigen for antibody therapy is that is should be abundant and homogeneous on the surface of tumor cells yet absent from normal tissues and adjacent noncancerous tissue (3). To identify potential targets for hepatocellular carcinoma, we screened for genes up-regulated in hepatocellular carcinoma by DNA microarray analysis (4). Microarray analysis has been applied to various aspects of cancer research, such as classification of cancer, elucidation of mechanisms of carcinogenesis, discovery of therapeutic targets, and the development of tumor markers (5–8). Recently, microarray studies on hepatocellular carcinoma presented gene lists, including a number of overexpressed genes (4, 9, 10).
The human homologue of the Drosophila Roundabout gene, ROBO1, encodes an axon guidance receptor that is defined as a novel subfamily of the immunoglobulin superfamily and is highly conserved from fruit flies to mammals (11–14). Drosophila robo functions as a gatekeeper controlling midline crossing (11, 13, 14). Human ROBO1 contains five immunoglobulin domains, three fibronectin III domains, a transmembrane domain, and an intracellular tail. ROBO1 is a member of the neural cell adhesion molecule family of receptors. On the other hand, ROBO1 was cloned as Deleted in U Twenty Twenty (DUTT1) from the lung cancer tumor suppressor gene region at 3p12. ROBO1/DUTT1 maps within a region of overlapping homozygous deletions seen in lung, breast, and kidney cancers (15). ROBO1 mRNA expression is silenced in patients with lung and breast cancer caused by hemizygous and homozygous deletions and/or hypermethylation of the promoter region of the ROBO1 gene. Therefore, ROBO1 is thought to be a candidate tumor suppressor gene.
In this study, we identified up-regulation of the ROBO1 gene in hepatocellular carcinoma by DNA microarray analysis and quantitative real-time PCR analysis. On immunohistochemical analysis, ROBO1 staining was observed in 83 of 98 cases of hepatocellular carcinoma (84.7%). In addition, the ectodomain of ROBO1 (soluble ROBO1) was detected in the culture medium of hepatocellular carcinoma cancer cell lines and in sera from hepatocellular carcinoma patients (6 of 11). This is the first report that ROBO1 is shed into serum in humans. We generated an anti-ROBO1 mAb (B2318C) with complement-dependent cytotoxicity against a ROBO1-expressing cell line and a liver cancer cell line PLC/PRF/5. Taken together, these results strongly suggest that ROBO1 represents a novel immunotherapeutic target and sensitive serologic marker for hepatocellular carcinoma.
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Materials and Methods |
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Cell lines. COS7, HEK293, and the hepatoblastoma cell line HepG2 were obtained from the American Type Culture Collection (Manassas, VA). The hepatocellular carcinoma cell lines HLE, HuH7, and PLC/PRF/5 and the hepatoblastoma cell line HuH6 were purchased from the Health Science Research Resource Bank (Osaka, Japan). An additional 14 tumor cell lines derived from different tumor types (stomach, lung, colon, brain, uterine cervix, and breast) were described previously (16).
RNA extraction and microarray analysis. Tissues or cells were lysed directly in Isogen reagent (Nippon Gene, Osaka, Japan) and homogenized. Total RNA was extracted according to manufacturer's instructions. Purchased RNA representing 30 different adult normal tissues, five different fetal tissues, and pooled total RNA from three samples each of moderately differentiated hepatocellular carcinoma, poorly differentiated hepatocellular carcinoma, and liver cirrhosis, along with total RNA from 19 cancer cell lines were analyzed using the HG-U133A array (Affymetrix, Santa Clara, CA) containing probes for 22,000 human genes. Further information on the source of RNA can be provided on request or is available at http://www.lsbm.org/db/index.html. Microarray analysis was done essentially as described previously (17, 18).
Quantitative real-time PCR. Tissues were lysed directly in Isogen reagent (Nippon Gene), and total RNA was extracted according to the manufacturer's instructions. Aliquots of 5 µg of total RNA were reverse transcribed into cDNA, using SuperScriptII (Invitrogen, San Diego, CA) with oligo-(dT) primers. Quantitative real-time PCR was done using SYBR Green I nucleic acid gel stain (BMA, Rockland, ME) with an iCycler iQ Detection System (Bio-Rad, Hercules, CA). Primers were as follows: 5'-GCATGCGATTGCAAATCTGA-3' and 5'-TTGAGACTGGAAGCAACGCAG-3' for ROBO1; 5'-AGAAGGAGATCACTGCCCTGGCACC-3' and 5'-CCTGCTTGCTGATCCACATCTGCTG-3' for ß-actin. PCR conditions were as follows: one cycle of 94°C for 3 minutes followed by 40 cycles of 94°C for 15 seconds, 63°C for 15 seconds, and 72°C for 30 seconds. The expression level of ROBO1 was first calculated as the relative ratio of ROBO1 to ß-actin in each sample.
Generation of anti-ROBO1 antibodies. We reported that foreign proteins can be displayed on the surface of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), and that mAbs can be produced by introducing the recombinant virus into mice in the absence of a protein purification step (19–21). Anti-ROBO1 antibodies were generated against various ROBO1 partial protein sequences using this system. The first immunoglobulin domain (Ig1; amino acids 22-113) and the third fibronectin III domain (Fn3; amino acids 738-855) of ROBO1 were amplified by PCR with the following primers: 5'-GGTACCCCTTCGTCAGGAAGATTTTCCAC-3' and 5'-GGTACCGAGTAATTCCTTGCTACACA-3' for Ig1; 5'-GGTACCCGCACCCAGTGCCCCACCCCAAGG-3' and 5'-GGTACCGCATCTGAAATCTGCTGAGCGAGG-3' for Fn3 and ligated into the gp64 gene. Culture media of Sf9 cells infected with recombinant baculoviruses encoding Ig1-BV and Fn3-BV were harvested and used as immunogens. Mouse mAbs (A7241A and B2318C) were then generated by the conventional method.
Immunoblotting analysis. The full-length ROBO1 cDNA was subcloned into the pcDNA3.1/V5-His TOPO TA vector (Invitrogen) by a PCR-based method using the ROBO1 primers 5'-ACCATGATTGCGGAGCCCGCTCAC-3' and 5'-GCTTTCAGTTTCCTCTAATTC-3' (ROBO1-V5/pcDNA3.1). ROBO1-V5/pcDNA3.1 was transiently transfected into COS7 cells or HEK293 cells using FuGENE 6 (Roche Diagnostic Systems, Inc., Branchburg, NJ) according to the manufacturer's instructions. Cells were lysed in 10 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 5 mmol/L EDTA, 1.0% Triton X-100, 1.0% sodium deoxycholate, 0.1% SDS, with protease inhibitor cocktail (Sigma, St. Louis, MO) and boiled for 10 minutes at 95°C with 0.1 mol/L 2-mercaptoethanol (Nacalai Tesque, Kyoto, Japan). Proteins were separated on 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Hybond P; Amersham Biosciences, Uppsala, Sweden). After blocking the membrane with 2% nonfat milk in TBS containing 0.05% Tween 20 for 1 hour, the membranes were incubated with A7241A (1.0 µg/mL), anti-V5 antibody (1:5,000; Invitrogen), or anti-ß-actin antibody (1:2,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as a primary antibody. Horseradish peroxidase–conjugated anti-mouse IgG antibody (1:50,000; Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a secondary antibody, and Enhanced Chemiluminescence PLUS Detection System (Amersham Biosciences) was used for chemiluminescent detection.
Immunohistochemical analysis. A total of 98 hepatocellular carcinoma specimens, including 15 well-differentiated, 69 moderately differentiated, and 14 poorly differentiated hepatocellular carcinomas, were prepared from the archives at the Tokyo University Hospital. Using a tissue microarrayer (Beecher Instruments, Silver Spring, MD), the area of interest in the donor paraffin block was cored twice with a needle 2 mm in diameter and transferred to the recipient paraffin block. Sections 4-µm thick were cut from the tissue microarray block and deparaffinized in xylene, washed in ethanol, and rehydrated in TBS. ROBO1 was stained with anti-ROBO1 mAb A7241A (20 µg/mL). Antigen retrieval was done in 10 mmol/L citrate buffer solution (pH 6) at 121°C for 10 minutes, and primary antibodies were applied for 1 hour followed by secondary staining with DAKO Envision+ Reagent (DAKO Ltd., Cambridge, United Kingdom) for A7241A and DAKO LSAB2 Reagent (DAKO) for the others. All sections were counterstained with Meyer's hematoxylin.
ROBO1 staining was scored according to the Clinical Trial Assay recommendations (0-3+) and was evaluated independently by two pathologists (J.S. and N.Y.). The results were compared, and any discrepancies were resolved by consensus at a meeting after further histopathologic reviews. The data were analyzed by the Fisher's exact test, and P < 0.01 was considered significant.
Establishment of ROBO1-expressing HEK293 cells. To establish ROBO1-V5 overexpressing HEK293 cell clones (ROBO1_HEK293), HEK293 cells were transfected with 1 µg of ROBO1-V5/pcDNA3.1 plasmid using FuGENE 6 (Roche Diagnostics, Indianapolis, IN). Cells were selected in the presence of 500 µg/mL Geneticin (Life Technologies, Rockville, MD) in DMEM supplemented with 10% fetal bovine serum. Expression of ROBO1 was analyzed by immunoblotting analysis using anti-V5 antibody (Invitrogen) and the mAb A7241A.
Flow cytometric analysis of ROBO1-expressing cells. ROBO1_HEK293 cells and liver cancer cell lines PLC/PRF/5, HepG2, HuH6, and HuH7 were washed with PBS and were resuspended in fluorescence-activated cell sorting buffer (PBS/1% bovine serum albumin/0.1% NaN3) at 
1 x 106 cells/mL. A total volume of 50 µL of cell suspension was incubated with 1 µg of the mAb B2318C for 60 minutes on ice, washed with fluorescence-activated cell sorting buffer, and incubated with 200 x FITC-labeled goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories) for another 30 minutes on ice. The cells were washed again, resuspended in fluorescence-activated cell sorting buffer, and analyzed on FACSCalibur (Becton Dickinson, Franklin Lakes, NJ).
Detection of the ectodomain of ROBO1 (soluble ROBO1) in sera of patients with hepatocellular carcinoma. The culture media of COS7 transiently expressing ROBO1 (ROBO1/COS7), HEK293 transiently expressing ROBO1 (ROBO1/HEK293), HepG2, PLC/PRF/5, HuH6, and HuH7 cells were analyzed by immunoblotting with the A7241A mAb (1 µg/mL). In addition, aliquots of 5 µL of 11 sera samples obtained from patients with hepatocellular carcinoma, three normal sera from healthy adults, five patients with chronic hepatitis, and five patients with liver cirrhosis were analyzed directly by immunoblotting analysis as described above. Culture media of PLC/PRF/5 cells and ROBO1/HEK293 were used as controls.
Complement-dependent cytotoxicity assay. Complement-dependent cytotoxicity activity was determined by calcein release assay (Wako Pure Chemical Industries, Osaka, Japan). Calcein-labeled cells were incubated with the mAb B2318C on ice for 15 minutes in DMEM supplemented with 10% fetal bovine serum. Baby rabbit complement (Cedarlane, Hornby, Ontario, Canada) was added at a final concentration of 25% and incubated at 37°C for 90 minutes. After incubation, 100 µL of supernatant was collected, and calcein concentration was measured by fluorometry with excitation and emission at 494 and 517 nm, respectively. Calcein concentrations in the supernatants of the cells incubated without complement and antibodies were considered spontaneous calcein release, and those of cells incubated in 1% NP40 solution were considered maximum calcein release. Cytotoxicity (%) was determined from the formula (A – C) / (B – C) x 100, where A, B, and C represent calcein release in each experiment, maximum calcein release, and spontaneous calcein release, respectively. All experiments were done in triplicate, and the figures show the means ± SD.
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Results |
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The ROBO1 gene expression pattern fulfilled all the desired characteristics. The ROBO1 gene was overexpressed in hepatocellular carcinoma specifically compared with other normal tissues and noncancerous (adjacent noncancerous liver tissues) except fetal tissues (Fig. 1A ). In addition, ROBO1 is a member of the neural cell adhesion molecule family of receptors and is a type I transmembrane molecule.
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Protein expression of ROBO1 in hepatocellular carcinoma. To characterize ROBO1 protein expression in hepatocellular carcinoma, we generated anti-ROBO1 mAbs using gp64-fused ROBO1 BV antigens. The mAb A7241A was generated by immunization with the Ig1-BV antigen and was used subsequently to detect ROBO1 protein by immunoblotting analysis and immunohistochemical analysis. When we analyzed the lysates of COS7 cells transfected with full-length cDNA of ROBO1 (ROBO1-V5/pcDNA3.1) with the mAb A7241A, we obtained the same results as with an anti-V5-tag antibody, which showed a band of 260 kDa specific to transfected cells (Fig. 2A
), suggesting that the mAb A7241A could specifically detect recombinant ROBO1 protein. Next, to confirm whether the mAb A7241A could detect endogenous ROBO1, cell lysates from 19 cancer cell lines (brain, lung, colon, stomach, liver, breast, and uterine cervix) were screened by immunoblotting analysis. The mAb A7241A detected ROBO1 protein only in cancer cell lines that showed high levels of ROBO1 mRNA expression by GeneChip analysis (Fig. 2B and C), suggesting that there is a correlation between ROBO1 mRNA and protein levels in cancer cell lines.
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Discussion |
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We showed that the mAb B2318C can kill both the liver cancer cell line PRC/PRF/5 and ROBO1-overexpressing HEK293 cells by complement-dependent cytotoxicity activity. We also confirmed that the mAb B2318C has antibody-dependent cell cytotoxicity against cells overexpressing ROBO1 (Supplementary Fig. S2). These observations suggest that ROBO1 is a promising target for antibody therapy in hepatocellular carcinoma.
The ligands of ROBO1 have been reported to be human homologues of Slit 1 to Slit 3, which play a vital role in axon guidance by signaling through Robo receptors (13, 14). Slit 2 is also a candidate tumor suppressor gene, which is silenced epigenetically in lung, breast, and colon cancers, and conditioned medium containing recombinant Slit 2 was shown to reduce growth of breast and colorectal cancer cells and to induce apoptosis in a colorectal cancer cell line (22, 23). ROBO1 is also a candidate tumor suppressor gene (15, 24). Therefore, the Slit-ROBO1 signaling pathway may be involved in the growth suppression signal in cancer cells. The expression of Slit 2 and also Slit 1 and Slit 3 in hepatocellular carcinoma is very low in poorly differentiated hepatocellular carcinoma (Supplementary Fig. S3); meanwhile, ROBO1 is overexpressed in hepatocellular carcinoma. Interestingly, mutations in the ROBO1 gene were not identified in liver cancer cell lines (data not shown), suggesting that ROBO1 may be overexpressed by negative feedback in hepatocellular carcinoma. Further studies to characterize the precise physiologic function of ROBO1 in hepatocellular carcinoma are currently under way in our laboratory.
In addition, we show that the ectodomain portion of ROBO1 is shed from the cell surface, and that this fragment of ROBO1 is present in the sera of patients with hepatocellular carcinoma but not in normal healthy adults or in patients with liver cirrhosis or chronic hepatitis. It is well established that the ectodomain of certain transmembrane molecules can be cleaved off by proteolysis, with the resulting shed fragment exerting important biological functions. Clearly, large-scale studies are needed to evaluate the potential of ROBO1 as a serologic marker for the early detection of hepatocellular carcinoma. We are currently pursuing the large-scale testing of an ELISA system capable of detecting ROBO1 in patient serum.
From a diagnostic perspective, shed ROBO1 may represent an excellent diagnostic marker for hepatocellular carcinoma. Yet, from a therapeutic perspective, it presents potential efficacy limitations to the use of ROBO1 as an antibody-based therapeutic to target tumor cells. It is of course possible that serum ROBO1 may accelerate the clearance of anti-ROBO1 therapeutic antibodies from the patient or impair the targeting of those antibodies to hepatocellular carcinoma tissues. Although, HER2, a well-known target for antibody therapy in breast cancer, is also a shed antigen, there is no significant correlation between shed antigen concentration and response status of patients treated with anti-HER2 antibody (25). Further experiments are needed to clarify the therapeutic potential of ROBO1 as an antibody-based therapy for the treatment of hepatocellular carcinoma.
This study is the first report showing that ROBO1 is expressed specifically at high levels in hepatocellular carcinoma, and that ROBO1 is shed from the membrane at a level detectable in the serum of patients with hepatocellular carcinoma. These results indicate that ROBO1 is a potential new serologic marker of hepatocellular carcinoma and may represent a new therapeutic target.
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Acknowledgments |
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
Received 12/20/05; revised 2/23/06; accepted 3/30/06.
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