CNTNAP4 signaling regulates osteosarcoma disease progression

Improved treatment strategies for sarcoma rely on clarification of the molecular mediators of disease progression. Recently, we identified the secreted glycoprotein NELL1 modulates osteosarcoma (OS) disease progression in part via altering the sarcomatous extracellular matrix and cell-ECM interactions. Of known NELL1 interactor proteins, Contactin-associated protein-like 4 (Cntnap4) encodes a member of the neurexin superfamily of transmembrane molecules best known for its presynaptic functions in the central nervous system. Here, CRISPR/Cas9 gene deletion of CNTNAP4 reduced OS tumor growth, sarcoma-associated angiogenesis, and pulmonary metastases. CNTNAP4 knockout (KO) in OS tumor cells largely phenocopied the effects of NELL1 KO, including reductions in sarcoma cell attachment, migration, and invasion. Moreover, CNTNAP4 KO cells were unresponsive to the effects of NELL1 treatment. Transcriptomic analysis combined with protein phosphoarrray demonstrated notable reductions in MAPK signaling with CNTNAP4 deletion, the ERK agonist isoproterenol restored OS cell functions among CNTNAP4 KO tumor cells. Finally, human primary cells and tissues in combination with publicly available sequencing data confirmed the significance of CNTNAP4 signaling in human sarcomas. In summary, our findings demonstrate the biological importance of NELL-1/CNTNAP4 signaling in disease progression of human sarcomas and suggest that targeting NELL-1/CNTNAP4 signaling represents a strategy with therapeutic benefit in sarcoma patients. Overall design: Bulk RNA sequencing of 143B human osteosarcoma cell line with or without CNTNAP4 gene deletion. 3 biological replicates for CNTNAP4 Vector Control (VC) and Knockout (KO) each