1437-1443 3/5/06 18:30 Page 1437 ONCOLOGY REPORTS 15: 1437-1443, 2006 Robo1/Robo4: Differential expression of angiogenic markers in colorectal cancer JÖRN GRÖNE1, OLIVER DOEBLER1, CHRISTOPH LODDENKEMPER2, BIRGIT HOTZ1, HEINZ-JOHANNES BUHR1 and SARAH BHARGAVA1 1Department of Surgery, 2Institute of Pathology, Charité - Medical School Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany Received December 6, 2005; Accepted February 9, 2006 Abstract. The family of roundabout (Robo) proteins is related Introduction to the transmembrane receptors and plays a major role in the process of axonal guidance in neurogenesis. It has recently The Robos (roundabouts) comprise a family of single-pass been shown that Robo proteins are also associated with transmembrane receptors identified in Drosophila, first tumor angiogenesis with Slit2 acting as the corresponding isolated in 1998. Four different Robos (Robo1, Robo2, Robo3, ligand. The aim of this study was to validate the differential Robo4) are described to date, whereas Robo1, 2 and 3 share expression by means of microarray analysis and real-time PCR structural homology containing five immunoglobulin (Ig) and to analyze the in situ expression of Robo1 and Robo4 in domains and three fibronectins in the extracellular region. In colorectal cancer. Quantitative analyses of Robo1, Robo4 and contrast, Robo4 is much smaller and consists of only three Igs Slit2 mRNA expression measured by large scale gene and two fibronectin. Robo4 is also termed magic roundabout. expression studies (Affymetrix U133A) showed a significant So far, human slits are considered the corresponding candidate up-regulation of Robo1 in tumor vs. normal tissue, whereas ligands for the Robo receptors. Slits are large secreted proteins Robo4 and Slit2 showed no significant deregulation. For and consist of a family of three genes, Slit1, Slit2 and Slit3. subsequent real-time PCR experiments, paired colorectal The Slit/Robo-interaction plays a critical role in neurogenesis tissue samples from cancerous and corresponding non- by mediating a repulsive signal during the process of axonal cancerous tissues were obtained from 50 colorectal cancer guidance. This signalling pathway has also been shown to be patients who underwent surgical resection. Robo1 mRNA important in myogenesis, inhibition of leukocyte migration and overexpression in cancerous tissue compared with normal the development of tumors (1-3). Robo/Slit has been detected counterparts was observed in 80% of the patients with a 4-fold in several tumor cell lines and in primary tumors such as expression in 45% and a 12-fold expression in 15%. For prostate carcinoma or breast cancer (3-5). It is indicated that Robo4, an up-regulation was detected in >70% (36/50). For Robo/Slit is involved in angiogenic mechanisms whereas Slit2, no differential expression was observed. The over- endothelial cells expressing Robo1 receptors are attracted by expression of Robo1 and Robo4 in tumor vs. normal tissue Slit2 which is secreted by human tumor cells (3). Moreover, was verified using real-time PCR. The histological analysis Wang and his co-workers successfully inhibited tumor growth revealed an expression of Robo1 mainly in tumor cells, by applying blocking antibodies into a xenograft animal model. whereas Robo4 is located primarily in endothelial cells of The expression of Robo4 is limited to the endothelium and has tumor vessels. Therefore, the Robo proteins provide potential been demonstrated to be strongly up-regulated in vessels of target structures for the anti-tumorigenic and anti-angiogenic tumors in the brain, colon and bladder (6). Further investigation therapy of colorectal carcinoma. revealed an essential role for Robo4 in in vivo angiogenesis (7). Thus, the Robo receptors seem to be a promising target for anti-tumorigenic and anti-angiogenic therapy, respectively. _________________________________________ For the assumed Robo receptor, Slit2, a suppressive role in colorectal cancer was assigned (8). Colorectal cancer (CRC) remains a significant cause of Correspondence to: Dr Jörn Gröne, Department of Surgery, Charité - Medical School Berlin, Campus Benjamin Franklin, cancer-related mortality in the Western world. As an alternative Hindenburgdamm 30, D-12200 Berlin, Germany treatment modality to conventional therapies, there has been E-mail: [email protected] an increasing interest in recent years in developing angio- suppressive agents. The inhibition of angiogenesis is based Key words: colorectal cancer, roundabout protein, Robo1, Robo4, on starving tumor cells of the blood supply which is required Slit2, angiogenesis, gene expression for tumor growth and represents a reasonable approach in the therapeutic treatment of cancer. Several anti-angiogenic agents alone, or in combination with conventional therapy modalities, are now in preclinical trials, such as protease inhibitors, 1437-1443 3/5/06 18:30 Page 1438 1438 GRÖNE et al: Slit/Robo IN COLORECTAL CANCER suppressors of angiogenic factors or direct inhibitors of step acquisition mode. Crossing points for each reaction were endothelial cell proliferation (9). defined by Second Derivative Maximum and arithmetic However, it has not been determined whether Robo1, baseline adjustment. Robo4 or Slit2 may also play a critical role in colorectal cancer or even in tumor angiogenesis. Therefore, our study sought to Immunohistochemistry for Robo1. Formalin-fixed, paraffin- examine the expression level of these target genes in colo- embedded sections (3 μm) were treated with 0.01% EDTA rectal cancer tissue and normal background tissue at the mRNA pH 8 in a microwave oven for 5 min at 800 W followed by level. Furthermore, we aimed to investigate the distribution 10 min at 200 W. Endogenous alkaline phosphatase activity of Robo1 and Robo4 in the cellular compartments of tissues. in sections was quenched by treatment with 1% hydrogen peroxide solution for 25 min. After blocking slides for 5 min Materials and methods (Powerblock, BioGenex), anti-Robo1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was applied in a 1:100 Patients and tissue preparation. Fifty patients with colorectal dilution and was incubated at 37˚C for 30 min. After cancer undergoing elective curative surgery from 2001 to 2003 thorough rinsing in PBS-Tween solution, slides were incubated at the Department of Surgery, Charité, Campus Benjamin with a 1:200 dilution of a biotinylated secondary anti-goat Franklin, Berlin, were prospectively recruited for this study antibody (Santa Cruz Biotechnology) for 20 min followed by with informed consent approved by the local ethics committee. a 20-min incubation with streptavidin peroxidase. For color Thirty out of 50 patients were UV-laser microdissected for development, slides were incubated for 5 min with the chiparray-based expression analysis. Tumor and normal tissue chromogen DAB (3.3'-diaminobenzidine tetrahydrochloride). from all 50 patients was macrodissected for RNA validation by All samples were lightly counterstained with hematoxylin for means of real-time PCR. 30 sec before dehydration and mounting. Immunostaining Microdissection. Laser microdissection of snap frozen was evaluated by two different observers [a clinician (O.D.) normal mucosa and corresponding primary colorectal carci- and a pathologist (C.L.)]. The estimated percentage of stained noma tissue specimens of 30 patients was performed as cells (%) and the average staining intensity of the positive previously described (10). For molecular analysis, ≤100,000 cells (normal and tumor) were expressed as follows: (0) none; cells were pooled. Tumor and normal cells were obtained from (+) weak; (++) intermediate; (+++) strong. different blocks from each case. Normal colorectal tissue was preferably taken from areas at the resection margin. In situ hybridization for Robo4. The Robo4 cDNA representing Macrodissection. Matched paired tissue samples from an the sequence from nucleotides 1252 to 1820 was amplified additional 20 CRC patients (a total of 50 patients) were cut by PCR using specific oligonucleotide primers: sense 5'-AG (cryo sections of 6-8 μm) and stained with hematoxylin and CCAACTGGACTGTAGTTG-3' and antisense 5'-GAGCC eosin (H&E). Tumor and normal tissue were verified by an ATAAAAAGTGCTGGTG-3' as described previously (12). experienced clinician and relevant areas were marked. Slides Human Robo4 cDNA was used as a template. The amplified (20 μm) were prepared and macrodissected with a sterile product was then subcloned into the pGEM cloning vector scalpel according to the mark. A new template H&E slide (Promega, Mannheim, Germany). The recombinant clones of 6-8 μm was prepared every 200 μm to control tumor and were isolated and sequenced to confirm the authenticity of normal cell areas. the sequence and to determine orientation. Plasmid DNA was linearized with PstI restriction endonuclease to transcribe sense RNA preparation and array hybridization. Poly A+ RNA pre- cRNA using T7 RNA polymerase and to transcribe antisense paration and hybridization of the Affymetrix human genome cRNA using SP6 RNA polymerase. The cRNA transcripts were U133 set (HG-U133) was performed as previously described digoxigenin-labeled by in vitro transcription using a DIG RNA (10). labeling kit (Roach Diagnostics Corp., Indianapolis, IN) according to the manufacturer's recommendations. Probes Quantitative real-time PCR. RNA was isolated using Nucleo- were then assessed for quality by agarose gel electrophoresis Spin® RNA II (Macherey-Nagel,