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.
Highlights
Neuroblastoma, an embryonic tumor of neuroectodermal origin, accounts for 11% of all cancerrelated deaths in children [1]
Time-resolved whole-genome expression www.impactjournals.com/oncotarget analyses on BE(2)-C cells transfected with empty- or GRHL1 vectors resulted in differential regulation over time of 170 genes, including CD9 [19]
This is of particular interest since widespread and extensive metastatic burden causes most neuroblastomarelated deaths, mirroring the perception that activating invasion and metastasis belongs to the cancer hallmarks acquired during the multistep development of human tumors [26]
Summary
Neuroblastoma, an embryonic tumor of neuroectodermal origin, accounts for 11% of all cancerrelated deaths in children [1]. Despite decades of considerable international efforts to improve outcome, long-term survival of high-risk disease remains as low as 20% [3]. Managing resistance to induction therapy, causing tumor progression and early death in ultrahighrisk patients, and managing chemotherapy-resistant relapses, which can occur years after initial diagnosis, remain major obstacles. MYCN amplifications [4, 5], telomerase activation by genomic rearrangements [6, 7], ATRX loss-of-function mutations or deletions [8,9,10] and germline or somatically acquired activating ALK mutations [11,12,13,14] define patient subgroups with highly aggressive and frequently therapy-resistant neuroblastomas. A commonality among these aggressive subgroups is that tumors undergo an as yet poorly understood invasionmetastasis cascade, which may be druggable to prevent increasing metastatic colonization
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