Activin A Suppresses Neuroblastoma Xenograft Tumor Growth via Antimitotic and Antiangiogenic Mechanisms Ekaterini Panopoulou, 1 Carol Murphy, 1,2 Heidi Rasmussen, 3 Eleni Bagli, 1 Einar K. Rofstad, 3 and Theodore Fotsis 1,2 1 Laboratory of Biological Chemistry, Medical School, University of Ioannina; 2 Biomedical Research Institute, Foundation for Research and Technology-Hellas, University Campus, Ioannina, Greece; and 3 The Norwegian Radium Hospital, Montebello, Oslo, Norway Abstract The tumor suppressor function of activin A, together with our findings that activin A is an inhibitor of angiogenesis, which is down-regulated by the N-MYC oncogene, prompted us to investigate in more detail its role in the malignant transfor- mation process of neuroblastomas. Indeed, neuroblastoma cells with restored activin A expression exhibited a diminished proliferation rate and formed smaller xenograft tumors with reduced vascularity, whereas lung metastasis rate remained unchanged. In agreement with the decreased vascularity of the xenograft tumors, activin A inhibited several crucial angio- genic responses of cultured endothelial cells, such as proteolytic activity, migration, and proliferation. Endothelial cell proliferation, activin A, or its constitutively active activin receptor-like kinase 4 receptor (ALK4T206D), increased the expression of CDKN1A (p21), CDKN2B (p15), and CDKN1B (p27) CDK inhibitors and down-regulated the expression of vascular endothelial growth factor receptor-2, the receptor of a key angiogenic factor in cancer. The constitutively active forms of SMAD2 and SMAD3 were both capable of inhibiting endothelial cell proliferation, whereas the dominant-negative forms of SMAD3 and SMAD4 released the inhibitory effect of activin A on endothelial cell proliferation by only 20%. Thus, the effects of activin A on endothelial cell proliferation seem to be conveyed via the ALK4/SMAD2-SMAD3 pathways, however, non-SMAD cascades may also contribute. These results provide novel information regarding the role of activin A in the malignant transformation process of neuroblastomas and the molecular mechanisms involved in regulating angiogenesis thereof. (Cancer Res 2005; 65(5): 1877-86) Introduction Several lines of evidence indicate that tumorigenesis in humans is a multistep process in which genetic alterations drive the progressive transformation of normal human cells into highly malignant derivatives. During this process, tumor cells acquire several capabilities that collectively dictate malignant growth (1). Sustained angiogenesis is one of the acquired capabilities of tumor cells supporting tumor mass growth and providing a gate for metastasis. Indeed, activation of oncogenes and inactivation of tumor suppressors alter the angiogenic switch by up-regulating angiogenic stimulators, like vascular endothelial growth factors (VEGF), and/or down-regulating angiogenic inhibitors such as thrombospondin-1 (2). Amplification of N-MYC oncogene is a frequent event in advanced stages (III and IV) of human neuroblastomas (3). N-MYC amplification correlates with poor prognosis (4) and enhanced vascularization (5) of human neuroblastomas, suggest- ing that N-MYC oncogene could activate tumor angiogenesis thereby enhancing neuroblastoma progression. Indeed, over- expression of N-MYC in a neuroblastoma cell line (SH-EP) resulted in an enhanced malignant phenotype of the trans- fectants (WAC2) allowing them to form well-vascularized tumors in nude mice (6). We have previously screened conditioned media from SH-EP007 and WAC2 cells and found that N-MYC overexpression in WAC2 cells down-regulated the expression of activin A, a protein that inhibited proliferation of cultured endothelial cell and angiogenesis in the chorioallantoic mem- brane assay (7). In this respect, activin A seemed to be an inhibitor of angiogenesis, a property that has not been previously ascribed to this molecule. Activin A, a homodimer of two inhibin hA subunits, is a member of the activin/inhibin family, which in turn belongs to the large transforming growth factor-h (TGF-h) superfamily of proteins (8). Activin A transduces its signals via binding to activin type II receptors, ActR-II, and ActR-IIB. The ligand/type II receptor complex then recruits, binds, and transphosphorylates the type I receptor, ActR-IB, also known as activin receptor-like kinase 4 (ALK4). Following activation of its kinase domain, ALK4 phosphorylates and activates selected members of a family of intracellular transducers known as SMADs, a process promoted by SARA-like proteins. On their way to the nucleus, the receptor activated SMADs (referred to as R-SMAD) associate with the related protein SMAD4. SMAD4 is referred to as the Co-SMAD, and is not a receptor substrate, but its presence is required for many of the gene responses induced by SMADs. Indeed, a specific activated SMAD complex accumulates in the nucleus, where it acts as a transcrip- tional regulator, controlling the expression of many genes (9). Activin and inhibin were originally isolated based on their activity to regulate follicle-stimulating hormone release from the anterior pituitary (10). However, activins have been found to exert a large spectrum of biological activities ranging from embryonic mesoderm induction to differentiation of hematopoietic cell lineages and repair processes in skin and brain (11–13). In cancer, activin A inhibits the proliferation of a variety of tumor (and normal) human cell types by blocking cell cycle progression from G 1 to S phase (14). Indeed, overexpression of activin A in human prostate cancer LNCaP cells inhibited proliferation, induced apoptosis, and decreased the tumorigenicity of these cells (15). Also, loss-of-function mutations of ALK4 have been identified in human pituitary tumors (16). As a consequence, activin A seems to function as a tumor suppressor, a property shared by the prototype molecule TGF-h, at least in early stages of tumorigenesis (17). Requests for reprints: Theodore Fotsis, Laboratory of Biological Chemistry, Medical School, University of Ioannina, Ioannina, Greece. Phone: 30-26510-97560; Fax: 30-26510-97868; E-mail: thfotsis@cc.uoi.gr. I2005 American Association for Cancer Research. www.aacrjournals.org 1877 Cancer Res 2005; 65: (5). March 1, 2005 Research Article Research. on August 9, 2015. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from