[CANCER RESEARCH 64, 8932– 8938, December 15, 2004] Dual Role of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 in Angiogenesis and Invasion of Human Urinary Bladder Cancer Leticia Oliveira-Ferrer, 1,2 Derya Tilki, 1 Gudrun Ziegeler, 1 Jessica Hauschild, 2 Sonja Loges, 3 Ster Irmak, 2 Ergin Kilic, 4 Hartwig Huland, 2 Martin Friedrich, 2 and Su ¨ leyman Ergu ¨n 1 1 Institute of Anatomy I and Departments of 2 Urology, 3 Oncology/Hematology, and 4 Pathology, University Hospital Hamburg-Eppendorf, Hamburg, Germany ABSTRACT Here, we show that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed in umbrella cells of bladder urothe- lium but is down-regulated in superficial bladder cancer, such as histo- logic tumor stage a (pTa) and transitional cell carcinoma in situ (pTis). Concurrently, CEACAM1 is up-regulated in the endothelia of adjacent angiogenic blood vessels. Mimicking the CEACAM1 down-regulation in the urothelium, CEACAM1 was silenced in bladder cancer cell lines 486p and RT4 using the small interfering RNA technique. CEACAM1 down- regulation was confirmed at the protein level by Western blot analyses. CEACAM1 silencing leads to a significant up-regulation of vascular en- dothelial growth factor (VEGF)-C and VEGF-D in quantitative reverse transcription-PCR. Correspondingly, supernatants from the CEACAM1- overexpressing bladder cancer cell lines reduce, but those from CEACAM1 silencing induce endothelial tube formation and potentiate the morphogenetic effects of VEGF. These data suggest that the epithelial down-regulation of CEACAM1 induces angiogenesis via increased expres- sion of VEGF-C and VEGF-D. Inversely, CEACAM1 is up-regulated in endothelial cells of angiogenic blood vessels. This in turn is involved in the switch from noninvasive and nonvascularized to invasive and vascularized bladder cancer. CEACAM1 appears to be a promising endothelial target for bladder cancer therapy. INTRODUCTION Angiogenesis is a prerequisite for tumor growth and metastasis and is regulated by angiogenic activators and inhibitors (1, 2). We recently showed that the human carcinoembryonic antigen-related cell adhe- sion molecule 1 (CEACAM1), formerly known as biliary glycoprotein or CD66a, exhibits angiogenic properties and functions as a morpho- genic effector for vascular endothelial growth factor (VEGF; refs. 3, 4). CEACAM1 is expressed in the newly formed immature blood vessels of different tumors and in new vessels formed during physi- ologic angiogenesis such as occur in wound healing and endometrial proliferation (3, 4). CEACAM1 is a member of the carcinoembryonic antigen family and can bind homophilically and heterophilically to the other CEA family members (5). Currently, 11 alternative splicing forms of the CEACAM1 gene are known (6, 7). CEACAM1 is expressed in epi- thelia and leukocytes in addition to endothelia. It has been shown that the mRNA expression of CEACAM1 is down-regulated in some tumors, such as colorectal and prostate carcinomas (8, 9). On the basis of such results, a tumor-suppressive role has been postulated. It recently was shown that CEACAM1 overexpression in the prostate cancer cell line DU-145 suppresses angiogenesis by mechanisms yet unknown (10). The increased expression of CEACAM1 in an exper- imental tumor model of human bladder cancer also suppressed tumor growth (11). It has been reported that the tumor inhibitory function of CEACAM1 depends on the cis-determinants in its cytoplasmic do- main (12). It was shown that CEACAM1 expression varies in prolif- erating and quiescent epithelial cells and that this influences their proliferation (13). The colocalization and interaction of CEACAM1 with paxillin and integrin  (3) recently were shown (14, 15). Most of these data have been obtained by studies of the membrane- bound CEACAM1 form. We showed that two soluble CEACAM1 forms at M r 120,000 and 50,000 are detectable in the conditioned media of endothelial cells after stimulation with VEGF and that these forms also exhibit angiogenic properties similar to CEACAM1 puri- fied from human granulocytes (3, 4). Concurrently, we showed that the capillaries of human bladder carcinoma also are positive for CEACAM1. On the basis of these results, we hypothesized that human bladder cancer serves as an appropriate model to study (1) whether CEACAM1 is involved in angiogenesis of bladder cancer and its expression in tumor vasculature depends on tumor stage; and (2) whether these parameters have a clinical relevance for the diagnosis and prognosis of bladder cancer. In this study, we show evidence that epithelial down-regulation but endothelial up-regulation of CEACAM1 activates angiogenesis in superficial noninvasive and nonvascularized urothelial tumors of bladder via increased expression of VEGF-C and VEGF-D. Accom- panied by that, CEACAM1 up-regulation in tumor adjacent blood vessels appears to correlate to the tumor switch from superficial to invasive. Strategies targeting endothelial CEACAM1 may be of ben- efit for antiangiogenic bladder cancer therapy. MATERIALS AND METHODS Tissue Samples. Normal tissue samples (n  7) from human bladder were obtained by biopsy, and tumor tissues of bladder cancer (n  38) were obtained from patients who had undergone surgical therapy. A part of the tissue pieces was fixed in 4% formaldehyde. The other part was fixed in Bouin’s solution and embedded in paraffin. In cases of cystitis (n  3), paraffin-embedded tissues were obtained from the Department of Pathology of the University Hospital Hamburg-Eppendorf. These tissues were sectioned and used for CEACAM1 immunohistochemistry. Cell Culture. Commercial human dermal microvascular endothelial cells (PromoCell, Heidelberg, Germany) were cultured on gelatin-coated dishes in endothelial cell growth medium MV (PromoCell) including 5% fetal calf serum (FCS). At confluence, they were used for endothelial tube formation assay. Human bladder cancer cell line 486p (16, 17) was grown in Roswell Park Memorial Institute 1640 medium (Life Technologies, Rockville, MD) with 15% FCS, 1% glutamine, and 1% penicillin/streptomycin (Life Technol- ogies). Human bladder cancer cell line RT4 was grown in McCoy’s medium (Life Technologies) with 10% FCS, 1% glutamine, and 1% penicillin/strepto- mycin (Life Technologies). Cells were cultured in six-well cluster dishes until 70% of confluency and then were used for transfection either for CEACAM1 overexpression or for CEACAM1 silencing. All of the cells were cultured at 37°C in 5% CO 2 /95% air. CEACAM1 Overexpression and Silencing in Bladder Cancer Cell Lines 486p and RT4. The cDNA encoding human full-length CEACAM1 was ligated into the plasmid pcDNA3.1/Hygro() (Clontech, Palo Alto, CA), which was designated pcDNA3.1/CEACAM1 and used for CEACAM1-over- expression studies. For CEACAM1 gene silencing, the target regions for the small interfering RNA (siRNA) sequences were selected from the cDNA of CEACAM1 according to the guidelines described by Elbashir et al. (18): Received 2/13/04; revised 8/25/04; accepted 10/1/04. Grant support: Deutsche Krebshilfe e.V. (German Cancer Foundation). 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. Requests for reprints: Suleyman Ergun, Department of Anatomy I, University Hospital Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. Phone: 49- 40-42803-4333; Fax: 49-40-42803-8416; E-mail: erguen@uke.uni-hamburg.de. ©2004 American Association for Cancer Research. 8932