Systemic Induction of the Angiogenesis Switch by the Tetraspanin D6.1A/CO-029
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Research Article Systemic Induction of the Angiogenesis Switch by the Tetraspanin D6.1A/CO-029 Sabine Gesierich,1 Igor Berezovskiy,1 Eduard Ryschich,2 and Margot Zo¨ller1,3 1Department of Tumor Progression and Immune Defence, German Cancer Research Centre; 2Department of Surgery, University of Heidelberg, Heidelberg; and 3Department of Applied Genetics, Faculty of Chemistry and Bioscience, University of Karlsruhe, Karlsruhe, Germany Abstract transcription of angiogenic factors (6, 7). Recent studies in Expression of the tetraspanin CO-029 is associated with poor knockout and transgenic mouse models have provided further prognosis in patients with gastrointestinal cancer. In a evidence that tumor angiogenesis is not only guided by the pancreatic tumor line, overexpression of the rat homologue, tumor cell itself, but is also closely tied to the tumor microenvi- ronment (8). D6.1A, induces lethally disseminated intravascular coagula- tion, suggesting D6.1A engagement in angiogenesis. D6.1A- Tetraspanins are a large family of proteins grouped according to overexpressing tumor cells induce the greatest amount of structural relatedness. The key feature of tetraspanins is their angiogenesis in vivo, and tumor cells as well as exosomes potential to associate with each other and with a multitude of derived thereof strikingly increase endothelial cell branching molecules from other protein families (9–11), the most prominent in vitro. Tumor cell–derived D6.1A stimulates angiogenic partners being integrins (12). The tetraspanin, D6.1A (rat)/CO-029 a h a h factor transcription, which includes increased matrix metal- (human), associates with 3 1 and 6 1 and, after disassembly of a h loproteinase and urokinase-type plasminogen activator se- hemidesmosomes, with 6 4. It also associates with the tetraspa- cretion, pronounced vascular endothelial growth factor nins, CD9 and CD81, and the immunoglobulin superfamily a expression in fibroblasts, vascular endothelial growth factor member, prostaglandin F2 receptor regulatory protein, a type II receptor expression, and strong D6.1A up-regulation in phosphoinositide-4-kinase, EpCAM, and CD44v4-v7 (13–15). sprouting endothelium. Thus, D6.1A initiates an angiogenic According to their association with different molecules, tetra- loop that, probably due to the abundance of D6.1A in tumor- spanins are assumed to function as adaptors that assemble protein derived exosomes, reaches organs distant from the tumor. complexes in defined membrane microdomains, called tetraspanin- Most importantly, because of the strong D6.1A up-regulation enriched microdomains, that provide a link to specific signal- on sprouting capillaries, angiogenesis could be completely transducing molecules (16). This mode of activity might explain inhibited by a D6.1A-specific antibody, irrespective of whether why tetraspanins are said to take part in a wide range of diverse or not the tumor expresses D6.1A. Tetraspanins have been functions (9, 11), such as B and T cell activation, platelet aggre- suggested to be involved in morphogenesis. This is the first gation, migration, proliferation (10, 17, 18), and tumor cell prog- report that a tetraspanin, CO-029/D6.1A, promotes tumor ression (18, 19). With respect to the latter, high expression of CD9 growth by its capacity to induce systemic angiogenesis that (20, 21) and CD82 (22) is mostly associated with a favorable can effectively, and with high selectivity for sprouting prognosis. CD151, D6.1A, and its human homologue, CO-029, are endothelium, be blocked by a D6.1A-specific antibody. (Cancer supposed to exert tumor-promoting activities (23–26). A possible Res 2006; 66(14): 7083-94) mechanistic basis for the prometastatic functions of tetraspanins could be their association with certain integrins and the accom- Introduction panying increase in cell motility (22, 23). Furthermore, tetraspanin interactions with platelets and leukocytes were suggested to The term angiogenesis defines the process of new capillary provide tumor cells with a survival advantage in the hostile formation from preexisting vasculature that occurs in physiologic environment which they encounter during metastatic spread (27). as well as pathologic conditions (1). Tumor cells essentially depend CD151 has also been reputed to be involved in cellular morpho- on angiogenesis to grow beyond a threshold size of a few cubic genesis, promoting the formation of cord-like structures, consid- millimeters (2). Angiogenesis is the result of an intricate balance ered as a preform of sprouting vessels (28). Finally, tumor cells can between proangiogenic and antiangiogenic factors, and the grow in mosaics with endothelial cells, and mosaic growth depends dominance of proangiogenic factors is called the angiogenic switch on tetraspanin expression (29). Taking into account that coagul- (3). One of the main angiogenic switch inducers is hypoxia (4), opathy and thrombosis are frequent complications, particularly in whereas the most well-known proangiogenic factor is vascular patients with lung and pancreatic cancer (30), it is important to endothelial growth factor (VEGF; ref. 5), and matrix metal- note that CD9, CD63, and CD151 are expressed by platelets. CD9 loproteinases (MMP) are essentially required to create space for and CD63 associate with aIIbh3, which supports activated platelet sprouting capillaries by degradation of the basal membrane as well adhesion to neutrophils and platelet aggregation (31). In CD151 as for the liberation of angiogenic factors and for inducing the knockout mice, platelet spreading is impaired, aggregation is reduced, and the bleeding time is prolonged. This is likely due to the absence of an outside-in signal provided by CD151, leading to Requests for reprints: Margot Zo¨ller, Department of Tumor Progression and Tumor Defence, German Cancer Research Centre, Im Neuenheimer Feld, 280 D-69120 the activation of aIIbh3 (32). Heidelberg, Germany. Phone: 49-6221-422454; Fax: 49-6221-424760; E-mail: m.zoeller@ D6.1A overexpression on a pancreatic rat adenocarcinoma line is dkfz.de. I2006 American Association for Cancer Research. associated with the formation of hemorrhagic ascites (15) and can doi:10.1158/0008-5472.CAN-06-0391 induce a severe consumption coagulopathy, such that rats become www.aacrjournals.org 7083 Cancer Res 2006; 66: (14). July 15, 2006 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research moribund due to disseminated intravascular coagulation rather supernatant after 2 days of culture by differential centrifugation. Super- than the tumor burden (13). These features prompted us to search natants were centrifuged twice for 10 minutes at 500  g, once for   for the possible involvement of D6.1A in angiogenesis. 20 minutes at 2,000 g, and once for 30 minutes at 10,000 g to eliminate cell debris. The purified supernatant was centrifuged for 90 minutes at 100,000  g using a SW41 rotor. The pellet was resuspended in PBS and Materials and Methods centrifuged again for 90 minutes at 100,000  g. Rats and tumors. BDX rats, bred at the animal facilities of the German Western blotting. Cell lysates (1% Triton X-100) were resolved on 12% or Cancer Research Centre, were kept under specific pathogen–free con- 15% SDS-PAGE under nonreducing conditions. Proteins were transferred ditions, fed sterilized food and water ad libitum. Rats were used for to Hybond enhanced chemiluminescence at 30 V overnight. After blocking experiments at the age of 6 to 10 weeks. BSp73AS (AS) cells, a pancreatic (5% fat-free milk powder), immunoblotting was done with the indicated adenocarcinoma line of the BDX rat strain (33), were transfected with D6.1A antibodies, followed by horseradish peroxidase–labeled secondary anti- cDNA (AS-D6.1A; ref. 13). For intravital microscopy, AS cells were bodies. Blots were developed with the enhanced chemiluminescence transfected with the enhanced green fluorescent protein (EGFP) cDNA– detection system. containing pEGFP-N1 vector (Becton Dickinson, Heidelberg, Germany). AS- Zymography. Cells (106) were seeded in 24-well plates (35). After D6.1A cells were transfected with the EGFP cDNA inserted in the overnight culture, cells were washed and starved in serum-free medium. pcDNA3.1Hygro vector (Invitrogen, Karlsruhe, Germany). Transfected lines Conditioned medium was collected after 24 hours and was centrifuged were selected in RPMI 1640, 10% FCS, and 500 Ag/mL of G418. The selection (15 minutes, 15,000  g) to remove cell debris. Aliquots were incubated with medium for double-transfected lines contained an additional 50 Ag/mL of Laemmli buffer (15 minutes, 37jC) and separated in a 10% acrylamide gel hygromycin. A BDX fibroblast line, generated by NiSO4 treatment, and containing 1 mg/mL of gelatin. The gel was washed for 30 minutes each in RAEC, a Wistar rat–derived aortic endothelial cell line (Cell-lining, Berlin, 2.5% Triton X-100, 2.5% Triton X-100/50 mmol/L Tris (pH7.5), and 2.5% A Germany) was maintained in RPMI 1640 and 10% FCS. Confluent cultures Triton X-100/50 mmol/L Tris (pH7.5), 5 mmol/L CaCl 2,1 mol/L ZnCl2. were trypsinized and split. Gels were incubated (24-48 hours, 37jC) in 50 mmol/L Tris (pH7.5), A Antibodies. The following monoclonal and polyclonal antibodies were 5 mmol/L CaCl2,1 mol/L ZnCl2, and stained with Coomassie blue. used: mouse anti-D6.1A (D6.1; ref. 34), mouse anti-a6h4 (B5.5; ref. 34), rabbit Matrigel assay. Tumor cells (105) were seeded on matrigel-coated anti-CD151 (14); anti-CD9