MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma

// Johannes Fabian 1,2 , Desiree Opitz 1 , Kristina Althoff 3 , Marco Lodrini 1,4 , Barbara Hero 5 , Ruth Volland 5 , Anneleen Beckers 6,7 , Katleen de Preter 6,7 , Anneleen Decock 6,7 , Nitin Patil 8 , Mohammed Abba 8 , Annette Kopp-Schneider 9 , Kathy Astrahantseff 4 , Jasmin Wunschel 1,4 , Sebastian Pfeil 4 , Maria Ercu 4 , Annette Kunkele 4 , Jamie Hu 1,10 , Theresa Thole 1,4 , Leonille Schweizer 11 , Gunhild Mechtersheimer 12 , Daniel Carter 13 , Belamy B. Cheung 13 , Odilia Popanda 14 , Andreas von Deimling 11,15 , Jan Koster 16 , Rogier Versteeg 16 , Manfred Schwab 17 , Glenn M. Marshall 13,18 , Frank Speleman 6,7 , Ulrike Erb 19 , Margot Zoeller 19 , Heike Allgayer 8 , Thorsten Simon 5 , Matthias Fischer 5,20,21 , Andreas E. Kulozik 22 , Angelika Eggert 4 , Olaf Witt 1,22 , Johannes H. Schulte 4 and Hedwig E. Deubzer 1,4,22,23 1 Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center (DKFZ) and German Consortium for Translational Cancer Research (DKTK), INF, Heidel­berg, Germany 2 Phenex Pharmaceuticals AG, Waldhofer Strase, Heidelberg, Germany 3 Department of Pediatric Hematology and Oncology, University Children’s Hospital Essen, Essen, Germany 4 Department of Pediatric Hematology, Oncology and SCT, Charite - University Hospital Berlin, Campus Virchow-Klinikum, Berlin, Germany 5 Department of Pediatric Hematology and Oncology, University of Cologne, Cologne, Ger­many 6 Center for Medical Genetics Ghent, Ghent University, De Pintelaan, Ghent, Belgium 7 Cancer Research Institute Ghent, De Pintelaan, Ghent, Belgium 8 Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, Centre for Biomedicine and Medical Technology, Mannheim, Germany 9 Department of Biostatistics, German Cancer Research Center (DKFZ), INF, Heidelberg, Germany 10 Yale University, New Haven, CT 11 Department of Neuropathology, University of Heidelberg, INF, Heidelberg, Germany 12 Department of Pathology, University of Heidelberg, INF, Heidelberg, Germany 13 Children’s Cancer Institute, UNSW, Randwick, NSW, Australia 14 Division of Epigenomics and Cancer Risk Factors, DKFZ, INF, Heidelberg, Germany 15 Clinical Cooperation Unit Neuropathology, DKFZ, INF, Heidelberg, Germany 16 Department of Oncogenomics and Emma Children’s Hospital, Academic Medical Center, University of Amster- dam, Meibergdreef, Amsterdam, the Netherlands 17 Neuroblastoma Genetics, DKFZ, INF, Heidelberg, Germany 18 Kids Cancer Centre, Sydney Children’s Hospital Randwick, Randwick, NSW, Australia 19 Experimental Surgery and Tumor Cell Biology, University of Heidelberg, INF, Heidelberg, Germany 20 Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany 21 Max Plank Institute for Metabolism Research, Cologne, Germany 22 Department of Pediatric Hematology and Oncology, Heidelberg University, INF, Heidelberg, Germany 23 Junior Neuroblastoma Research Group, Experimental and Clinical Research Center of the Max-Delbruck Center for Molecular Medicine in the Helmholtz Community and the Charite - University Medicine Berlin, Lindenberger Weg, Berlin, Germany Correspondence to: Hedwig E. Deubzer, email: // Keywords : antimetastatic therapy, chromatin modulation, histone deacetylases, grainyhead-like transcription factor family, tetraspanin family Received : April 29, 2016 Accepted : August 24, 2016 Published : August 27, 2016 Abstract The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma.